Thesis and Pharmacological
THE SYNTHESIS AND PHARMACOLOGICAL
STUDIES ON A NOVEL ANTIMICROBIAL AGENT &
NEW NICKEL CATALYSTS AND THEIR
APPLICATIONS IN ORGANIC SYNTHESIS
Nikki Ying-Tung Man BSc (Hon.) | BMus
Supervisors: Dr. Scott Stewart Dr. Katherine Hammer
Prof. Allan McKinley Prof. Thomas Riley
This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia School of Molecular Sciences School of Biomedical Sciences
Chemistry Microbiology
2017 ii |
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THESIS DECLARATION
I, Nikki Man, certify that:
This thesis has been substantially accomplished during enrolment in the degree.
This thesis does not contain material which has been accepted for the award of any other degree or diploma in my name, in any university or other tertiary institution.
No part of this work will, in the future, be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint-award of this degree.
This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text.
The work(s) are not in any way a violation or infringement of any copyright, trademark, patent, or other rights whatsoever of any person.
Technical assistance was kindly provided by Dr. Mark Howard for the NMR studies of protein/ligand interaction that is described in Section 2.3.2; Dr. Gavin Knott, Dr. Amanda Lewis and Dr. Jason Schmidberger assisted in protein crystallography and X-ray data collection (Section 2.3.1); genome mapping and analysis of bacteria was performed by PhD candidate Daniel R. Knight (Section 3.4.5); and fluorescent microscopy imaging and data processing was performed by Associate Professor Paul Rigby (Section 4.5.2)
This thesis contains published work and/or work prepared for publication, some of which has been co-authored.
Signatur
Date: 28t July 2017
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ABSTRACT
Part I of this thesis describes the synthesis and pharmacological evaluation of antibacterial compound 135C (I). Through tandem Mizoroki–Heck reactions and changes at synthetic handles, six new analogues were synthesised in generally reasonable yields for the study of their interaction with protein. Due to solubility limitations of the analogues, the interaction between protein serum albumin and 135C was alternatively confirmed by way of saturation transfer NMR experiments.
A variety of parameters were determined for the biological evaluation of 135C, including its spectrum of activity against pathogenic bacteria, its mutagenic, haemolytic, and resistance profiles. A comparison of the whole genome sequences of the generated mutants and their wildtype counterparts revealed a change in teichoic acid-associated tag genes. Leakage, lysis and time-kill studies were performed, along with investigations into any synergistic or antagonistic interaction with six different antibiotics.
In addition, a 135C analogue bearing an azide moiety was successfully prepared for a copper-free interaction with a cyclooctyne fluorescent probe in an attempt to elucidate the molecular mode of action of I.
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Part II of this thesis describes the use of new air-stable nickel(0) complexes as catalysts in cross-coupling reactions. Catalytic cross-coupling processes usually depend on the use of expensive palladium compounds or air-sensitive nickel(0) complexes. In this thesis, nickel(0) phosphite complexes were trialled for their ability to catalyse the C–C bond forming Mizoroki–Heck and Suzuki–Miyaura cross-coupling reactions.
Some of these novel complexes (II) proved moderately successful as catalysts in Mizoroki–Heck reactions between aryl triflates and vinyl ethers, and very proficient in Suzuki–Miyaura cross-coupling reactions between aryl boronic acids and aryl tosylates. vi |
TABLE OF CONTENTS
Thesis Declaration …………………………………………………………...... iii Abstract ……………………………………………………………………………... iv Table of Contents ……………………….………………………………………….... vi Acknowledgements ………..………………………………………………………...... x Abbreviations ………………………….………………………………………….... xii Authorship declarations ………………………………………………………….... xv
PART I: The synthesis and pharmacological studies on a novel antimicrobial agent Chapter 1: General Introduction...... 2 1.1 Antibiotics in the pre-antibiotic era ...... 2
1.2 Establishment of the golden age of antibiotics ...... 2
1.3 What is antimicrobial resistance? ...... 7
1.4 Antimicrobial resistance: current crisis...... 7
1.5 Antibiotic resistance in hospitals and communities ...... 9
1.5.1 MRSA in Western Australia ...... 10
1.6 New antimicrobial agents ...... 11
1.7 Approaches to addressing the antibiotic crisis ...... 15
1.8 Project background and origins...... 17
1.9 Project aims ...... 21
Chapter 2: Synthesis, Bioisosteres and Protein Interaction ...... 22 2.1 Synthesis of 135C ...... 22
2.1.1 Discussion of the spectrometric data of 67 and crystallographic data of 52 .. 27
2.2 Bioisosteric analogues of 135C ...... 30
2.2.1 Sulfonic acid analogue ...... 32
2.2.2 Sulfonamide analogue ...... 33
2.2.3 Tetrazole analogues...... 35
2.2.4 Miscellaneous analogues ...... 40
2.2.5 Remarks on analogue syntheses ...... 40
2.3 Interaction with serum albumin ...... 41 | vii
2.3.1 Protein crystal structure...... 41
2.3.2 NMR studies of 135C/BSA interaction ...... 43
Chapter 3: Biological Evaluations of 135C ...... 48 3.1 Brief introduction ...... 48
3.2 In vitro susceptibility tests ...... 48
3.2.1 Spectrum of activity ...... 48
3.2.2 Broth microdilution assay ...... 49
3.2.3 Susceptibility of Gram-positive bacteria ...... 49
3.2.4 Susceptibility of anaerobic bacteria ...... 51
3.2.5 Susceptibility of Gram-negative bacteria ...... 51
3.2.6 Antimicrobial activity of analogues ...... 53
3.3 Toxicology studies ...... 54
3.3.1 The Ames Salmonella/mutagenicity tests ...... 54
3.3.2 Haemolytic activity of 135C ...... 55
3.4 Antimicrobial resistance studies ...... 56
3.4.1 Serial passage of S. aureus isolates with compound 135C ...... 56
3.4.2 Stability of resistance ...... 57
3.4.3 Cross-resistance studies of generated mutants ...... 58
3.4.4 Growth fitness of generated mutants...... 58
3.4.5 Genomic comparison of generated mutants and wildtypes...... 61
3.5 Final remarks on the biological evaluation of 135C ...... 66
Chapter 4: Investigations into the Modes of Action of 135C ...... 68 4.1 Mode of action overview ...... 68
4.2 Leakage and lysis experiments...... 68
4.2.1 Cell lysis ...... 68
4.2.2 Cell leakage ...... 69
4.3 Time-kill studies ...... 69
4.4 Synergy experiments ...... 70 viii |
4.5 Azide-tagged compound for confocal microscopy ...... 72
4.5.1 Synthesis of azide analogue of 135C ...... 73
4.5.2 Fluorescent microscopy of azide-tagged 135C with S. aureus NCTC 6571 . 82
Chapter 5: Summary and Conclusions- Part I ...... 88 Chapter 6: General Introduction- Part II...... 94 6.1 The Importance of the cross-coupling method in organic synthesis ...... 94
6.2 Notable breakthroughs in metal-catalysed cross-coupling reactions ...... 95
6.2.1 Humble beginnings ...... 95
6.2.2 Discovery phase of catalytic cross-coupling reactions ...... 96
6.2.3 The rise of palladium in cross-coupling chemistry ...... 98
6.2.4 Improvements on palladium-catalysed processes ...... 99
6.2.5 Other notable key cross-coupling examples ...... 101
6.3 Recent pharmaceutical applications ...... 102
6.4 General mechanism of cross-coupling reactions ...... 103
6.5 Organonickel chemistry ...... 105
6.5.1 Historical perspectives and notable discoveries ...... 105
6.5.2 Nickel vs. Palladium ...... 108
6.6 Project aims ...... 113
6.6.1 Catalytic activity of air-stable Ni(0) complexes ...... 114
6.6.2 Summary of project aims ...... 115
Chapter 7: Mizoroki–Heck Cross-Coupling Reactions ...... 116 7.1 Mizoroki–Heck reaction of electron-rich olefins with nickel(0) phosphites ...... 116
7.2 Mechanism of the Mizoroki–Heck cross-coupling reaction ...... 118
7.3 Optimisation of the nickel phosphite catalysed Mizoroki–Heck reaction ...... 121
7.3.1 Nickel phosphite catalysed Mizoroki–Heck reaction of 225 ...... 125
7.4 Scope of the nickel phosphite catalysed Mizoroki–Heck reaction ...... 126
7.4.1 Reactivity of phenylhalides and pseudohalides ...... 126
7.4.2 Scope of the aryl triflate coupling partner with butyl vinylether ...... 127 | ix
7.4.2 Application of nickel(0) phosphite system on the synthesis of compound 135C ...... 128
Chapter 8: Suzuki–Miyaura Cross-Coupling Reactions ...... 130 8.1 Suzuki cross-coupling reactions ...... 130
8.2 Mechanism of the Suzuki–Miyaura cross-coupling reaction...... 131
8.3 Suzuki–Miyaura cross-coupling reaction of aryl pseudohalides & boronic acid...... 132
8.4 Optimisation Studies ...... 133
8.5 Scope of aryl tosylate coupling partner with phenylboronic acid...... 138
8.6 Scope of the aryl boronic acid coupling partner with 4-cyanophenyltosylate. ... 140
Chapter 9: Summary and conclusions- Part II ...... 143 Chapter 10: Experimentals ...... 146 10.1 Chemistry general protocol ...... 146
10.2 Instruments and materials ...... 146
10.2.1 Chemistry ...... 146
10.2.2 Microbiology ...... 147
10.3 Experimental procedures- Part I...... 148
10.3.1 Biological assays ...... 148
10.3.2 Protein Crystallography ...... 153
10.3.3 Saturation Transfer Difference NMR...... 154
10.3.4 Synthesis ...... 155
10.3.5 Confocal microscopy ...... 174
10.4 Experimental procedures- Part II ...... 174
10.4.1 General procedure for Mizoroki–Heck cross-coupling reactions ...... 174
10.4.2 General procedure for Suzuki–Miyaura cross-coupling reactions ...... 177
10.4.3 General procedure for the synthesis of aryl tosylates ...... 186
Appendices ...... 193 Appendix A- Crystallographic data of compound 135C ...... 193
Bibliography ...... 196 x |
ACKNOWLEDGEMENTS
First I’d like to thank you, Scott Stewart, for all your patience, encouragement and advice throughout my PhD, and without whom I most probably wouldn’t have pursued this challenging journey over the past 4 years. I’m very grateful for all that you have done for me and thank you so much for your friendship and support.
To Allan, your kindness and caring spirit really inspired me to partake in the representation of the welfare of fellow students and to be involved in university administration and politics. Dipping my toes into this unknown territory has been a very eye-opening experience and I’ve learnt so much from it! I also really value the times that we’ve done the chemistry magic show together, they really have been the highlights of my time here at UWA!
Boss Lady Kate, you’re awesome. Thanks so much for taking me on as your PhD student even though I had no microbiology background. Your guidance has been invaluable and I really appreciate our chats about everything random. And dear Thomas, thanks for your expertise on all things micro, poo, toxins, lunches, coffees, fun facts about cars, and enjoyable opera tracks.
Many thanks to Lindsay, Mark and Gareth for all your help with NMR and our jolly chats! Thanks to Paul and Alysia for your help with the confocal work- I was pretty excited about your special cameras! Thanks to Dan for helping me out with the genome sequencing, even though you’re super busy finishing your own PhD!!
To Charlie, Valerie, Gavin, Amanda and Jason, thanks for all your help on protein crystallography. It was super interesting and fun to see you remotely shoot beams at midnight at crystals! Of course, thank you to all the lab techs for your technical assistance and good chats, especially Oscar and Paul.
One thing that made a huge impact throughout my PhD journey was the amount of outreach and teaching that I was lucky enough to be involved in. Thank you to the now dissembled SPICE team for the educational fun times in the outback. Also I’d like to thank Dino for being an excellent chemistry unit coordinator. I honestly think that you | xi are one of the best people who have come along and made the student experience, school and our teaching better than what it used to be.
To the wonderous Stewart, Koutsantonis and Riley labs, you have made doing research so enjoyable. You really are the best people to work with because you make the labs such wonderful environments to be in. Special thanks to Sven, you pretty much taught me everything in the chemistry lab alongside Scott. Thanks to Louisa for your generosity and infectious happiness. As I write this, I realise that I will write a novel if I thanked everyone who helped me throughout the past 4 (somestimes struggletown) years, where it be about actual work or just by bringing joy into my life with your presence. But I do have to mention and thank Jeremy, Dennis, Matthys, Kieran, Siobhan, Becky, David, Chuck, Campbell, Jackson, Lien, and our recently lost friend Ming.
Last but not least, to my beautiful friends and family, your love and encouragement has been so important to me, not just for doing science, but for living life. Thank you so much for being part of it.
Also, if I wasn’t this chatty, I think I would’ve finished this doctorate business a long time ago…AAAND this research was supported by an Australian Government Research Training Program (RTP) Scholarship!!
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ABBREVIATIONS Ac acetyl Alk alkyl APCI atmospheric-pressure chemical ionisation Ar aryl b.p. boiling point 2,2’-bis(diphenylphosphino)-1,1’-binaphthyl (when part of a BINAP (binap) complex) 2,2'-bis(dibenzo[d,f][1,3,2]dioxaphosphepin-6-yloxy)-1,1'-biph BiPhenOPhos enyl Boc tert-butoxycarbonyl Bu n-butyl C. albicans Candida albicans C. difficile Clostridium difficile cat. catalytic CDT (cdt) 1,5,9-cyclododecatriene (when part of a complex) COD (cod) 1,5-cyclooctadiene (when part of a complex) COSY correlation spectroscopy COT (cot) 1,3,5,7-cyclooctatetraene (when part of a complex) Cp cyclopentadienyl Cy cyclohexyl
Cy2NMe N, N-dicyclohexylamine d days DavePhos 2-dicyclohexylphosphino-2’-(N,N-dimethylamino)biphenyl DBU 1,8-Diazabicycloundec-7-ene DCM dichloromethane DCyPF 1,1’-bis(dicyclohexylphosphino)ferrocene DIBO 4-dibenzocyclooctynol DIPEA N,N-diisopropylethylamine DiPrPF 1,1’-bis(diisopropylphosphino)ferrocene DMAP N,N-dimethyl-4-aminopyridine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DPPE (dppe) 1,2-bis(diphenylphosphino)ethane (when part of a complex) 1,1’-bis(diphenylphosphino)ferrocene (when part of a DPPF (dppf) complex) DPPPE 1,5-bis(diphenylphosphino)pentane DtBuPF 1,1’-bis(di-tert-butylphosphino)ferrocene E. coli Escherichia coli e.g. exempli gratia (from Latin meaning ‘for example’) EDG electron-donating group EI electron ionisation equiv. equivalent ESBL extended-spectrum β-lactamase ESI electrospray ionisation et al. et alii (from latin meaning “and others”) | xiii
Et ethyl etc. et cetera (from Latin meaning ‘and the rest’) ether diethyl ether EWG electron-withdrawing group FDA Food and Drug Administration FQRP Fluoroquinolone-resistant Pseudomonas aeruginosa GC gas chromatography h hours HMDS hexamethyldisilazane, bis(trimethylsilyl)amine HPLC high-performance liquid chromatography HRMS high resolution mass spectrometry HSA human serum albumin Hz hertz i.e. id est (from Latin meaning ‘that is’) i-Pr isopropyl IR infrared (spectroscopy) IUPAC International Union of Pure and Applied Chemistry LPS lipopolysaccharide m meta M molar m multiplet M. catarrhalis Moraxella catarrhalis m.p. melting point MBC minimum bactericidal concentration MDR multidrug resistant Me methyl Mes mesityl MIC minimum inhibitory concentration min minutes mMRSA multi-resistant MRSA mRNA messenger ribonucleic acid MRSA methicillin-resistant Staphylococcus aureus MS mass spectrometry MscL large-conductance mechanosensitive ion channel NaHDMS sodium hexamethyldisalazide NDA new drug applications NMR nuclear magnetic resonance NOE nuclear Overhauser effect o ortho p para Ph phenyl Piv pivalate pKa acid dissociation constant ppm parts per million Pr n-propyl xiv |
PRSP penicillin-resistant Streptococcus pneumoniae Ps. aeruginosa Pseudomonas aeruginosa PVL Panton-Valentine leucocidin r.t. room temperature rac racemic
Rf retention factor RT room temperature s singlet s seconds S. aureus Staphylococcus aureus S. capitis Staphylococcus capitis S. epidermidis Staphylococcus epidermidis S. haemolyticus Staphylococcus haemolyticus S. hominis Staphylococcus hominis S. pneumoniae Streptococcus pneumoniae S. pyogenes Streptococcus pyogenes S. saprophyticus Staphylococcus saprophyticus S. warneri Staphylococcus warneri SAR structure activity relationship SDW sterilised distilled water stoich. stoichiometric t tertiary t triplet TBS tert-butyldimethylsilyl Temp. temperature TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TMEDA N,N,N’,N’-tetramethylethane-1,2-diamine Tol tolyl TPM triphenyl methyl Ts tosyl, p-toluenesulfonyl UV-vis ultraviolet visible VISA vancomycin-intermediate S. aureus VRE vancomycin-resistant Enterococci Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene XPhos 2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl
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AUTHORSHIP DECLARATION: CO-AUTHORED PUBLICATIONS This thesis contains work that has been published and/or prepared for publication.
Details of the work: Boulos, R. A., Man, N. Y. T., Lengkeek, N. A., Hammer, K. A., Foster, N. F., Stemberger, N. A., Skelton, B. W., Wong, P. Y., Martinac, B., Riley, T. V., McKinley, A. J. & Stewart, S. G. (2013). Inspiration from Old Dyes: Tris (stilbene) Compounds as Potent Gram‐Positive Antibacterial Agents.Chemistry-A European Journal, 19(52), 17980-17988 Location in thesis: Part I Student contribution to work: 40 %
Details of the work: Kampmann, S. S., Man, N. Y. T., McKinley, A. J., Koutsantonis, G. A., & Stewart, S. G. (2016). Exploring the Catalytic Reactivity of Nickel Phosphine–Phosphite Complexes. Australian Journal of Chemistry, 68(12), 1842-1853. Location in thesis: Chapter 7 Student contribution to work: 10 %
Details of the work: Man, N. Y. T., Knight, D. R., McKinley, A. J., Stewart, S. G., Riley, T. V., Hammer, K. A. Investigating the spectrum of antibacterial activity and mode of action of a novel stilbene antibacterial agent, prepared for publication. Location in thesis: Chapter 3, 4.2-4.4 Student contribution to work: 90 %
Student signature: Date: 28th July 20
I, Allan McKinley, certify that the student statements regarding their contribution to each of the works listed above are correct
Coordinating supervisor signature: Date: xvi |
PART I: SYNTHESIS AND PHARMACOLOGICAL STUDIES ON A NOVEL ANTIMICROBIAL AGENT 2 | Chapter 1: General Introduction
Chapter 1: General Introduction 1.1 Antibiotics in the pre-antibiotic era Antimicrobial agents are one of the most successful chemotherapies in medical history. Prior to the so-called “antibiotic era” beginning with the discovery of penicillin in 1928, evidence of the use of antibiotics in different ancient cultures has been well documented. These occurrences include tetracycline detection in human skeletal remains from both ancient Sudanese Nubia dated back to 350 – 550 CE,1 as well as the Egyptian Dakhleh Oasis from the late Roman period.2 Historical anecdotal evidence has also been collected regarding the use of the red soil in Jordan for the treatment of skin infections, which was recently confirmed to contain antibiotic-producing bacteria that can kill Micrococcus luteus and Staphylcoccus aureus.3 Antimicrobial activity has also been found in a number of traditional Chinese medicines that have been used for thousands of years throughout the Asian continent and beyond.4
1.2 Establishment of the golden age of antibiotics Contrary to popular belief, penicillin was not the first antimicrobial drug. The first two clinically useful antimicrobials in human medicine were fully synthetic compounds that originated from the synthetic dye industry; both originated from Béchamp’s economically viable synthesis of atoxyl (1), also known as arsanilic acid, via the reduction of nitrobenzene in the presence of iron and HCl, which he published in 1854.5
Whilst working on the development of synthetic dyes as treatment for African sleeping sickness, Paul Erhlich and co-workers synthesised around 2000 compounds based on the structure of arsanilic acid at the beginning of the 20th century.6 From this study, “Compound 606” (2) was found to be effective against Treponema pallidum, the bacterium responsible for syphilis (Figure 1-1). In 1910, this organoarsenic compound was marketed as Salvarsan, and it became the most commonly prescribed drug in the world as well as the first antimicrobial agent available for clinical use.7 It is interesting to note that the original structure (2) assigned by the inventors is incorrect; in 2005,
Ronimus and co-workers determined that Salvarsan is a mixture of the cyclo-As3 (3) 8 and cyclo-As5 (4) compounds. Unfortunately, Salvarsan led to severe detrimental side effects such as deafness, and is difficult to administer due to its low water-solubility.7 In 1912, a more water soluble, but less efficacious derivative with lower toxicity was launched on the market as Neosalvarsan (5).9 1.2 Establishment of the golden age of antibiotics | 3
Figure 1-1. Structures of atoxyl (1) and Salvarsan (2- incorrectly assigned structure, 3 and 4- revised structures).
The second class of clinically used antimicrobial agents were sulfa drugs. Specifically synthesised by Otto,10 the first synthetic azo dye chrysoidine (Figure 1-2, 6), was shown to be bactericidal in vitro against streptococci.11 Motivated by this discovery and their expertise in synthetic dyes, IG Farben/Bayer chemists Fritz Mietzsch and Joseph Klarer synthesised over 300 azo dye compounds for biological testing between 1927 and 1932.12 From this library of compounds, Gerhard Domagk was able to determine the excellent therapeutic properties of 7, a red dye (later named Protonsil), against streptococci in in vivo assays.13 In the same year that protonsil (7) was launched commercially (1935), a study by scientists at the Paris Pasteur Institute revealed that protonsil (7) is actually a prodrug that is metabolised to the authentic active therapeutic, a sulfanilamide.14 In the following decade, more than 5000 analogues of sulfanilamide were synthesised, from which a number became effective therapeutics.15
Figure 1-2. Structures of chrysoidine (6) and the synthesis of protonsil (7) via sulfanilamide (8).11,16
The year 1928 saw the beginning of one of the most important scientific breakthroughs in clinical medicine- Alexander Fleming’s rediscovery of penicillin (Figure 1-3, 9), a 4 | Chapter 1: General Introduction secondary metabolite of the Penicillium chrysogenum fungus.17 In the following years, Fleming was not able to draw much interest from chemists to help resolve problems in the purification and isolation of the active substance from the bacterial residue. However, in 1940, Ernst Chain and Howard Florey published a culture and purification process that was able to yield sufficient quantities of penicillin for clinical testing.18 This naturally-derived antimicrobial agent was deemed an excellent antimicrobial candidate in relation to its safety and efficacy, and it reduced the mortality rate caused by bacterial infections from 10 – 12 % in WWI down to 3 % in WWII.19 The three seminal discoveries of the first classes of antimicrobial agents set the standards for future research in drug discovery. For the next 20 years, new antimicrobial classes emerged one after the other, leading to “a golden age of antimicrobial chemotherapy”20 (Figure 1-4).
Figure 1-3. Structure of Penicillin G (9)
1970: 1908: Salvarsan 1952: Erythromycin (macrolide) Cephalexin (arsenical) 1932: Protonsil 1955: Vancomycin (glycopeptide) 1990: Linezolid (sulfonamide) 1957: Rifamycins (ansamycin) (cephalosporin) (oxazolidinone)
1986: Fluoroquinolone 1942: Penicillin (β-lactam) 1961: Trimethoprim (pyrimidine) (norfloxacin) 1944: Streptomycin (aminoglycoside) 1962: Naladixic acid (quinolone), 1945: Chlortetracycline (tetracycline) Virginiamycin (streptogramin) 1949: Chloramphenicol (phenylpropanoid), Neomycin & Gentamicin (aminoglycosides)
Figure 1-4. Timeline of antimicrobial class discovery.21,22 Brackets indicate structural class of drugs.
Many new antimicrobial compounds were derived from natural sources similar to penicillin; chlortetracycline (10) was first isolated from the actinomycete Streptomyces aureofaciens,23 and streptomycin (11) from the soil microbe Streptomyces griseus.24 Other classes were first discovered and commercially launched via chemical synthesis, such as nalidixic acid (12),25 and chloramphenicol (13)26 (Figure 1-5). 1.2 Establishment of the golden age of antibiotics | 5
Figure 1-5. Structures of chlortetracycline (tetracycline, 10), streptomycin (aminoglycoside, 11), nalidixic acid (quinoline, 12), and chloramphenicol (13).
Antimicrobial agents can also be classified by their core chemical structures, which in many instances relates to their mode(s) of action (Figure 1-6). * The five main antimicrobial modes of action and examples include: o Inhibitors of cell wall synthesis such as β-lactams (penicillin, 9) and glycopeptides (vancomycin, 14); o Inhibitors of cell membrane function such as polymyxins (colistin, 15); o Inhibitors of protein synthesis such as aminoglycosides (streptomycin, 11), oxazolidinones (linezolid, 16), macrolides (erythromycin, 17), chloramphenicol (13) and tetracyclines (chlortetracycline, 10); o Inhibitors of DNA and RNA synthesis such as fluoroquinolones (ciprofloxacin, 18), metronidazole (19) and rifampin (20) o Inhibitors of other metabolic processes such as sulfonamides (protonsil, 7), nitrofurans (nitrofurantoin, 21) and trimethoprim (22).27
* The mode of action of an antimicrobial compound is the means by which it inhibits (bacteristatic) or kills bacteria (bactericidal). Bactericidal agents usually kill microorganisms by interfering with the development of either cell walls or cytoplasm contents of the bacterial cell. Bacteriostatic agents typically inhibit the growth of bacteria by interfering with metabolic processes such as protein synthesis.28 6 | Chapter 1: General Introduction
Cell wall synthesis and cell membrane function inhibitors
Protein synthesis inhibitors
DNA and RNA synthesis inhibitors
Inhibitors of other metabolic processes
Figure 1-6. Chemical structures of antimicrobial agents as classified by their mode(s) of action. 1.3 What is antimicro bial resistance? | 7
1.3 What is antimicrobial resistance? A resistant bacterial strain is a microorganism that has acquired defence against a particular antimicrobial agent to which it was originally susceptible.28 When the susceptibility of an organism to an antimicrobial compound has decreased to the extent where the agent is no longer clinically useful, the bacteria has acquired ‘clinical resistance’.28
Resistance to antimicrobial agents can result from the disruption of one or more of the essential steps within the mode of action of the antimicrobial agent; this includes processes such as altering the cell’s physiology or structure.28 There are two different ways in which bacteria can achieve resistance; either by environmental or self-mediated means. Resistance that is a direct consequence of the physical or chemical characteristic of the surrounding environment is called environmentally-mediated resistance; this type of resistance results from alterations of the structure of the drug itself or from a positive change within the organism’s normal physiological response to the drug.29
Resistance that is due to alterations in the genetics of the bacteria is called microorganism-mediated resistance; susceptibility tests are used to detect this type of resistance in vitro. Within this category, resistance is further categorised as intrinsic or acquired. Intrinsic resistance results from the normal genetic, structural or physiologic state of the bacteria. Acquired resistance is caused by the genetic alteration of the organism’s normal cellular physiology and structure, including genetic mutations and/or acquisition of foreign genes.28
1.4 Antimicrobial resistance: current crisis Antimicrobial resistance throughout history can be traced back to well before the antibiotic era by phylogenetic reconstructions.30,31 However, it is only in the latter half of the 20th century that this previously natural phenomenon has become a problem due to its acceleration following the use of antimicrobials in a clinical setting. In the case of penicillin, Fleming was one of the first to suggest the potential for resistance, warning that the dosage period and concentration of the drug was important to preserve its activity.32 In 1940, observations by Abraham and Chain suggested that bacteria could destroy penicillin by enzymatic degradation.33 Ultimately in 1947, the first case of penicillin-resistant S. aureus was reported,34 followed by the detection of erythromycin- resistant staphylococci in Japan, England, France, and the USA in 1955. Eventually, 8 | Chapter 1: General Introduction methicillin-resistant Staphylococcus aureus (MRSA), one of today’s most prominent resistant pathogens, emerged in 1961 in the UK.21 Increasing numbers of resistant pathogens were reported over the next decades, peaking in the 1990s, where many common pathogens are now multi-drug resistant (MDR) (Figure 1-7).21
1961: MRSA 1967: Penicillin- 1982: Cephalosporin-resistant MRSA resistant Neisseria 1983: Penicillin-resistant Enterococcus faecium 1947: Penicillin- gonorrhoeae and 1987: Vancomycin-resistant Enterococci resistant S. aureus Streptococcus pneumoniae
Early-1990s: MDR Pseudomonas aeruginosa 1955: Erythromycin- Mid-1990s: MDR Mycobacterium tuberculosis resistant staphylococci Mid-late 1990s: most MRSA resistant to fluoroquinolones 1997: Vancomycin-intermediate S. aureus (VISA) 1999: Linezolid-resistant Enterococcis Early 2000s: Community-acquired (CA)-MRSA 2003: Linezolid-resistant S. aureus
Figure 1-7. Timeline of first cases of reported antibiotic-resistance.20–22
Antimicrobial resistance is one of the most problematic public health issues that our society is facing today. As an example, the prevalence of MRSA has increased from 5% in 1980 to 50% of documented S. aureus infections in 2000 (Figure 1-8).35 This rise of MDR pathogens is placing significant pressure on essential health care systems around the world36 as infections caused by these pathogenic bacteria are causing greater levels of morbidity and mortality than in the past.37 As an example, estimates indicate that in 2008, approximately 2000 patients died from infectious diseases in Australia.38 Therefore, effective antibiotic therapy remains a critical component of preventing deaths caused by infectious diseases.
1.5 Antibiotic resistance in hospitals and communities | 9
MRSA = Methicillin-resistant Staphylococcus aureus; VRE = Vancomycin-resistant Enterococcus; FQRP = Fluoroquinolone-resistant Pseudomonas aeruginosa Figure 1-8. Trends in the incidence of multi-drug resistant bacteria.35
Due to the increasing trend of antimicrobial resistance, the number of currently available treatment choices for more serious bacterial infections is steadily decreasing.37 A number of pan-resistant†39,40 bacterial infections are seen as untreatable and their occurrence is being observed in both community and hospital environments.37 In August 2016, a notification of carbapenem-resistant Enterobacteriaceae (CRE) was reported from a patient in Nevada;41 the isolate was determined to be a strain of Klebsiella pneumoniae and was resistant to 26 antibiotics. This recent case highlights the impending future of multidrug-resistant pathogens in both hospitals and communities.
1.5 Antibiotic resistance in hospitals and communities MDR pathogens were previously only a hospital problem; however, this resistance has diffused into the community due to many factors, including the extensive use and commercialisation of antimicrobial agents in everyday life.42–44 Since the late 1990s, the main multi-resistant pathogens that have been widespread and more lethal are MRSA, vancomycin-resistant Enterococcus (VRE), extended-spectrum β-lactamase (ESBL)-
† Pan-resistant is defined as “pathogens that are specifically resistant to 7 antimicrobial agents (cefepime, ceftazidime, imipenem, meropenem, piperacillin-tazobactam, ciprofloxacin, and levofloxacin)”.11 However, as Pan- is a Greek prefix meaning “all”, the term “pan-resistant” may be interpreted to mean resistant to all antimicrobials. An example of a pan-resistant pathogen is Acinetobacter baumannii, a Gram-negative bacterium that is distinguished by its rapid development of resistance to the majority of antimicrobials, including aminoglycosides, fluoroquinolones, and carbapenems.12
10 | Chapter 1: General Introductio n producing Gram-negatives, and penicillin-resistant Gram-positive strains such as and penicillin-resistant Streptococcus pneumoniae (PRSP).45,46
At the same time, there was also extensive use of antimicrobial agents that affected the gut flora such as fluoroquinolones and other broad-spectrum agents.45 These antimicrobial-mediated changes in the gut flora have led to new problems such as infection with Clostridium difficile, which causes life-threatening medical conditions from infectious diarrhoea to pseudomembranous colitis. The emergence of a plasmid- mediated ribosomal-based resistance mechanism in Staph. aureus and Staph. epidermidis has also been reported, along with outbreaks of linezolid-resistant enterococci and linezolid-resistant MRSA.36 These MDR bacteria increase the risks involved in routine surgical procedures8 such as hip replacements. The elderly and immunocompromised patients are particularly susceptible to such infections.45
1.5.1 MRSA in Western Australia An example of a pathogen that has moved from hospitals to communities is MRSA. This pathogen was first reported in Australia in 1968.47 In WA, a screening and control policy was implemented after a hospital outbreak epidemic in 1982; as a direct result of this policy, multi-resistant MRSA (mMRSA) did not establish in WA when it became endemic in eastern Australian hospitals in the late 1980s and early 1990s.48 Notifications of MRSA have increased steadily from 99 in 1983 to 2318 cases in 2002 as shown in a study by Riley and co-workers.49 In this study, instances of hospital- acquired MRSA were relatively low compared to community-acquired cases (Figure 1- 9). This low hospital:community-acquired ratio remains into the 2000s, as the overall incidences of MRSA steadily increases.50 From the latter set of data (Figure 1-10), the community-acquired strains were also classified into Panton-Valentine leucocidin (PVL)‡-positive and PVL-negative strains; PVL-positive isolates have higher virulence and hence pose a greater threat to the community.50
‡ PVL is a β-pore-forming cytotoxin that leads to neutrophil lysis. The toxin is present in most community acquired MRSA genes, apart from strains found in Australia.475 | 11
Figure 1-9. Notifications of hospital-acquired (HA) and community-acquired (CA) MRSA in Western Australia (WA), 1983–2002.49
HA=healthcare-associated strains; CA= community-associated strains; PVL=Panton-Valentine leukocidin Figure 1-10. Notifications of MRSA in Western Australia, 2004 -2012.50
1.6 New antimicrobial agents The number of new antimicrobial drugs that are released to consumers has been steadily decreasing since the end of the golden age of antibiotics (Figure 1-4). This is, to some extent, due to the low investment of pharmaceutical companies into the antimicrobial drug research and development pipeline.36 However, it is more largely due to the increasingly stringent processes and regulations that new drugs have to pass from the point of their discovery to their release into the pharmaceutical market.37,45 This approval process is now more difficult to clear through regulatory agencies than ever 12 | Chapter 1: General Introduction before. Pharmaceutical companies and the Food and Drug Administration (FDA) have raised their expectations on the adverse side effects of drugs but at the same time, do not provide any “clinical trial guidelines” for researchers to follow.45 This ambiguity surrounding the expected safety and efficacy criteria of an acceptable drug deters many developers of antimicrobial agents as it is complicated, time consuming and costly for companies to support.37,45
Between the early 1940s and 1960s, a large number of new classes of antimicrobial agents were developed and launched commercially (Figure 1-4),21,22 whereas the following 40 years saw only three new drug classes on the pharmaceutical market. Furthermore, of the 14 new antimicrobial drugs that have been approved and launched on the market since the turn of the century (Figure 1-11), only three contain novel structural classes (Figure 1-12; daptomycin (lipopeptide, 23), retapamulin (pleuromutilin, 24) and fidaxomicin (macrocycle, 25)), with the remainder containing structures based on existing classes. This situation is concerning as analogues of existing drugs have encountered cross-resistance issues in the past.36 Additionally, 26 new antibiotics that were approved by the FDA between 1980 and 1999 were subsequently withdrawn or discontinued by 2009;51 most of the withdrawals were due to the lower efficacy of the drug compared to related analogues already on the market.
Daptomycin (lipopeptide)* Telavancin Ceftolozane (cephalosporin) (glycopeptide) Fidaxomicin Ceftazidime-avibactam Tigecycline (macrocycle) (cephalosporin/β-lactamase (tetracycline) * inhibitor)
Retapamulin pleuromutilin)* Ceftaroline fosamil Tedizolid (oxazolidinone) Doripenem (carbapenem) (cephalosporin) Oritavancin (glycopeptide) Dalbavancin (lipoglycopeptide) Ceftolozane + tazobactam (cephalosporin/β-lactamase inhibitor)
Figure 1-11. FDA approved drugs since 2003. Brackets indicate structural class of drugs. * indicates new drug classes.
1.6 New antimicrobial agents | 13
Figure 1-12. Structures of daptomycin (23), retapamulin (25) and fidaxomicin (24).
As of September 2016, there are 39 drugs in the current U.S.A. antimicrobial clinical development pipeline;52 two have been submitted as new drug applications (NDA), 13 in Phase I,§ 13 in Phase II** and 11 in Phase III†† clinical trials. Overall, 12 of the 39 drugs contain core chemical structures that have not formerly been comprehensively used as an antimicrobial in human medicine. Eleven new antimicrobial agents are in their late stages of development and will be available on the market soon if they pass all the necessary protocols (Figure 1-13). These include Cadazolid53 (oxazolidinone- quinolone hybrid, 26), Cefiderocol54 (cephalosporin, 27), Iclaprim55 (diaminopyrimidine, 28), β-lactam/β-lactamase inhibitor combination therapy of imipenem (29a) /cilastatin (29b) + relebactam (29c) (MK-7655),56 Plazomicin57 (aminoglycoside, 30), Taksta‡‡58,59 (fusidic acid), Zabofloxacin60 (fluoroquinolone, 31), and tetracyclines Omadacycline61 (32) and Eravacycline62 (33).52
§ Phase I = Testing of drug on a small groups of people (<100) for safety evaluation (dosage range and side effects) ** Phase II = Testing of drug on a large group of people to assess efficacy and safety †† Phase III = Testing of drug on large groups of people to further assess efficacy, effectiveness and safety ‡‡ Structure not available. Proprietary oral formulation of fusidic acid owned by Cempra Pharmaceuticals 14 | Chapter 1: General Introduction
Figure 1-13. Current antimicrobial therapies in phase III clinical trials.52
As antimicrobial agents are likely to face potential cross-resistance issues if their chemical structures are based upon already existing drugs, it is important to develop new classes of antimicrobial agents. Currently, there are two such compounds in the development pipeline which have completed Phase 1 and Phase 2 clinical trials (Figure 1-14). The first is Carbavance (meropenem (34a) + vaborbactam (34b)), a combination of β-lactam and β-lactamase inhibitor developed by Rempex Pharmaceuticals Inc.; although the carbapenem is of the already existing β-lactam class, the cyclic boronate structure of the β-lactamase inhibitor is a novel structure. The other therapeutic of a 1 . 7 A pproaches to addressing the antibiotic crisis | 15 novel class is Lefamulin (BC-3781) (35), a pleuromutilin developed by Nabriva Therapeutics AG.
Figure 1-14. Current antimicrobial therapies of novel classes in phase III clinical trials
1.7 Approaches to addressing the antibiotic crisis Originally only observed in a clinical microbiology setting, the current antibiotic resistance crisis is now a complex problem faced by our society. The prevention of resistance and the search for new therapeutics requires effort not only from the scientists, but also from health care professionals, and the agricultural and pharmaceutical industries. As prevention always takes precedence over cure,63,64 the education of the general public, policy makers and governmental legislative bodies is also vital to addressing the antibiotic resistance problem.32,65
From a scientific stance, a number of different approaches are being taken to address the issue, one of which is the improvement of existing therapeutics. This approach was first implemented as early as the mid-1940s, utilising the method of chemical semisynthesis.15 One of the first applications of antimicrobial semisynthesis involved the structural modifications of naturally occurring aminoglycosides (concurrently by scientists at Merck and Park, Davis & Co.) and tetracyclines (by Lloyl Conover at Pfizer). The platinum-catalysed reduction of an aldehyde group on streptomycin (36) produced dihydrostreptomycin (37);66 catalytic hydrodehalogenation of chlorotetracycline (38) over Pd/C produced tetracycline (39),67 one of the most effective and widely used antimicrobials (Figure 1-15). These seminal innovations enabled improvements in the search for more therapeutic options (both fully and semi- synthetically), and are still extensively used today.
16 | Chapter 1: General Introduction
Figure 1-15. Antimicrobial semisynthesis of dihydrostreptomycin (37) and tetracycline (39).
Although new antimicrobial agents can be made via the improvement of currently available therapeutics, these types of agents may face a higher risk of cross-resistance development due to their similar structures.68 It is therefore imperative to search for novel classes of compounds that may have a longer enduring clinical lifespan.
Structural modification processes have also led to hybrid antimicrobials. This approach has not been very fruitful since many hybridised compounds are less effective and not advantageous over the simple combination of the individual class structures.69 Examples of such cross-modified antimicrobials are quinalactams70 (a fluoroquinolone- cephalosporin hybrid), and rifamycin-fluoroquinolone hybrids.71 Although clinically unsuccessful, the synthesis of these hybrids is interesting from a chemistry perspective, in that the latter hybrid (Figure 1-16; CBR-2092, 40) contains a heterobicyclic chiral linker between the two components (Figure 1-16).72 Nevertheless, some hybrid antimicrobials have been successfully used in a clinical setting, such as the oxazolidone- fluoroquinolone hybrid, Cadazolid (Figure 1-13, 26),53 which is currently in phase III clinical trials.52
| 17
Figure 1-16. Structure of rifamycin-fluoroquinolone hybrid, CBR-2092 (40).
From a historical perspective, natural products have been an abundant source of antimicrobial therapeutics, with a large number of clinically successful drugs derived from soil actinomycetes.73 As such, the search for natural products as new leads is an ongoing pursuit. Other approaches for the search of novel antimicrobial classes include screening of structurally diverse antimicrobial peptides,§§ as well as targeting resistance mechanisms, and strategically targeting new bacterial cellular processes or structures as modes of action.
1.8 Project background and origins Naturally-occurring stilbenes *** are known for their diverse bioactivities74 including anti-cancer (phoyunbenes (41),75 halophilol A (42)76 and combretastatins (43)77), anti- diabetic (rumexoid(44))78 and antimalarial (stilbenes isolated from Artocarpus integer (45))a8 activity. Many stilbene derivatives have also shown activity against bacteria,80 including combretastatin B5 (46), a natural product isolated from Combretum woodii leaves81 (Figure 1-17).
§§ These “host-defence” peptides are released by all eukaryotes as an immune response to infections.
*** Stilbenes have the diarylethene structure: 18 | Chapter 1: General Introduction
Figure 1-17. Structures of Naturally-occurring stilbenes and derivatives.
In 2011, the Stewart and McKinley research groups at UWA conducted an in silico study for a compound which would affect the physical cellular structure of bacteria by interrupting the large-conductance mechanosensitive ion channel (MscL). This study was based on the molecular templates of a range of commercial dyes with reported antimicrobial properties. Specifically, the in silico study modelled upon the spatial arrangement of the phenyl ring systems in triphenyl methyl (TPM) dyes, including brilliant green (47), methyl violets (48) and aluminon (49) (Figure 1-18).
Figure 1-18. Antimicrobial dyes and simple aromatic compounds with reported antimicrobial properties
Brilliant green (47) has been reported to display antimicrobial activity against Gram- positive bacteria,82–85 and crystal violet (48c) has been used as both an antimicrobial agent and a histological stain for years.84–87 The protonated equivalent of Aluminon, aurintricarboxylic acid, has been shown be active against Escherichia coli as a 1.8 Project background and origins | 19 topoisomerase II inhibitor as well as by disrupting mRNA functions.88–91 Similar structures with several carboxylic acid groups (50) have also been reported to have activity against Gram-positive bacteria.92 Based on these structures and functional groups, this study led to the discovery of stilbene compound 135B (Figure 1- 19, 50), which was later synthesised in Figure 1-19. Structure of Compound 135B. the laboratory.93
Although 135B was found to be active in a number of antimicrobial assays, later experiments concluded that the minimum inhibitory concentration (MIC) ††† was increased by at least 32-fold in the presence of horse serum.94 Specifically, 135B had an MIC of 2 μg/mL against S. aureus NCTC 6571; however, in the presence of 5% horse serum the MIC was increased to 32 μg/mL. This result suggested that tris-acid 135B, given its additional low solubility in aqueous environments, was unlikely to be effective in vivo.
In reported examples, a reduced efficacy of compounds containing carboxylic acid motifs has been linked to human serum albumin (HSA) binding which proved problematic for eventual clinical trials and ruled out many drug candidates.95,96 Other benzoic acid-based drugs also have a high incidence of HSA binding, for example ibuprofen and warfarin.95,97 To increase the potential for 135B to be used in in vivo assays, several techniques of combating the serum albumin binding through drug formulation were considered. For example, by using serum albumin as a drug carrier,98 or by using cyclodextrins as a drug delivery tool in a similar fashion to other antibiotics (cefotiam and itraconazole).99 In the end, a structural modification was chosen because of the ease of synthesis of 135B. Several aspects were considered in designing analogues of the lead compound, including the spatial orientation of the acid groups, the degree of saturation of the molecule and introducing other functional groups that would perform as isosteres of the terminal benzoic acids.
††† MIC is the lowest concentration of drug that inhibits microbial growth. 135B is named so because the substitution pattern of the core aromatic ring is in the 1, 3 and 5 positions. 20 | Chapter 1: General Introduction
During this study, the compound 135C (Figure 1-20, 52) was conceived following a comparison with an anthranilic acid-derived class of antimicrobial agents. In these examples, it was suggested that the pKa of the carboxylic acid functional group plays a role in Figure 1-20. Structure of Compound 135C the potential for interaction with HSA, where the lower the pKa of the acid, the more protein the compound bound.100,101 Compound 135C, with a methylene spacer between the tri-styryl groups and carboxylic acids, has a computationally determined pKa of 4.34. This is less acidic than that of compound 135B (pKa = 4.08) bearing benzoic acid moieties.
The synthesis of tris-acid 135C was first completed in 201294 and follows a simple process from commercially available and halogenated starting materials. The latter stages of this synthesis followed that of the synthesis of 135B. This relatively short process incorporates a tandem Mizoroki–Heck cross-coupling reaction to attach each of the pendant arms to the central aromatic core; such tandem cross-coupling reactions require less solvents and separation aids.102 This approach makes the compound more economical to produce if larger scale production is required, and with less environmental impact. Currently, standard step-wise syntheses are employed for many antimicrobial agents on the pharmaceutical market.
Furthermore, this relatively simple molecule contains no chiral centres, adding to its ease of production and purification. From a medicinal chemistry perspective, initiating a structure-activity-relationship (SAR) study with easily synthesised molecules is more likely to proceed smoothly, and a library of analogues more accessible and can be prepared on a reasonable scale. It was hoped that through these structural modifications, the potential improvement of activity as well as changes in physical properties would enhance the capability of this class of molecules as pharmaceutical agents.
1.9 Project aims | 21
This structurally simple synthetic compound was found to exhibit antimicrobial activity against many Gram-positive bacteria, including MRSA, at concentrations comparable to existing antibiotics and better than its precursor 135B.103,104 However, when tested in the presence of 5% horse serum, the MIC against S. aureus NCTC 6571 was 2 μg/mL compared to 0.125 μg/mL in the absence of serum. This 16-fold increase in MIC suggested that activity was also affected by serum, possibly in a manner similar to that of compound 135B.
This reference compound contains a triacid functionality and a tri-styrene motif, representing a structural class not seen in the current antimicrobial literature; other natural stilbenes with antimicrobial activity, such as resveratrol105 have been isolated but none contain this tri-styrene motif. Thus, given that this compound was outside any existing antimicrobial classes, the advantage of developing it as a potential antimicrobial agent was increased since there may have been a reduced likelihood of antimicrobial cross-resistance106.
1.9 Project aims The overarching aim of this research was to further investigate the antimicrobial activity of compound 135C and the synthesis of bioisosteric analogues. Specific objectives of the project included: - synthesis of bioisosteric analogues via carboxylic acid group replacement, - determining the spectrum of activity against a wide range of organisms, - establishing its cytotoxicity and mutagenicity profiles, and; - conducting investigations to understand the mode of action of 135C. 22 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction
Chapter 2: Synthesis, Bioisosteres and Protein Interaction 2.1 Synthesis of 135C The first synthesis of 135C was performed in 2012 as a part of an honours research project.94 The synthesis involved a key Mizoroki–Heck cross-coupling reaction between commercially-available 1,3,5-tribromobenzene (53) with methyl 2-(4- vinylphenyl)acetate (54) (See Scheme 2-5). The original synthetic pathway of the precursor 54 began with a Grignard reaction of 4-vinylbenzylchloride (55) and CO2 to obtain the carboxylic acid (56) (Scheme 2-1).107 This transformation was capricious as the Grignard intermediate (57) requires a carbon dioxide treatment to form a magnesium carboxylate complex (58), followed by an acidification to acquire the final product (56). The yields obtained through this reaction ranged from 10 to 36 %, depending on the
CO2 quenching and physical mixing techniques used. Although classic Grignard reactions are generally high yielding, conversions of alkyl,108 and aryl chlorides have been reported to be low-yielding unless the reaction is performed with additional catalysts109,110 or via electrolysis.111
Scheme 2-1. Grignard transformation of 4-vinylbenzylchloride (55) to 2-(4- vinylphenyl)acetic acid (56).
In order to find a more efficient and higher yielding synthesis, a literature search identified a pathway that provides access to the carboxylic acid from 4-vinylbenzyl chloride 55 (Scheme 2-2).112 Treating the benzyl chloride 55 with KCN and 18-crown-6 allows the formation of a crown ether/potassium ion complex ion, allowing the cyanide ion to more efficiently undergo an SN2 reaction with the alkyl halide to provide 4- vinylbenzylnitrile (59) in 82% yield.113 The spectral data for this compound matched those reported in the literature.114
2 . 1 S y n t h e sis of 135C | 23
Scheme 2-2. Two-step synthesis from 4-vinylbenzylchloride (55) to 2-(4- vinylphenyl)acetic acid (56) via 4-vinylbenzylnitrile (59).112
With the nitrile 59 in hand, a second step involving its hydrolysis with potassium hydroxide afforded the carboxylic acid 56 in 90% yield.112 The identity of 2-(4- vinylphenyl)acetic acid 56 was immediately confirmed by the downfield shift of the methylene proton signals from 3.59 ppm to 3.64 in the 1H NMR spectra, as well in the carboxylate carbon at 176.73 ppm in the 13C NMR spectra, matching those reported in the literature.114 Although this synthetic pathway involves an extra step, it is more reliable than the CO2 quench process previously used, as well as consistently providing a higher overall yield of 74%.
The methylation of the carboxylic acid 56 to ester 54 (Scheme 2-3) was achieved upon treatment with methyl iodide (81 % yield).107 The appearance of a singlet at 3.69 ppm and 52.1 ppm in the 1H and 13C NMR spectrum, respectively, confirmed the structure of the compound along with the comparison to literature spectra.107 A dimethylsulfate- promoted methylation was also trialled,112 however, its slightly lower yield (78 %) and longer reaction time made it the less favourable reagent. Due to its high levels of toxicity, the use of dimethylsulfate is also strictly controlled in Australia and therefore is more inconvenient to use.
Conditions and reagents: a) MeI (1.3 equiv.), DBU (1.3 equiv.), THF, 0 - 20 oC, 3 h, 81%; or b) o Me2SO4 (1 equiv.), DBU (1.2 equiv.), MeOH, 0 - 20 C, 12 h, 78%.
Scheme 2-3. Two methylating conditions of 2-(4-vinylphenyl)acetic acid (56) to methyl ester (54).
24 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction
With the methylated carboxylic acid styrene 54 in hand, the key and tandem reaction could be attempted. In this process, three consecutive Mizoroki–Heck cross-coupling reactions were carried out, allowing the formation of three new carbon-carbon bonds, each of these between an alkyl halide and an alkene precursor. The Mizoroki–Heck cross-coupling reaction like others in this class is based on the fundamental catalytic chemistry of palladium in its two dominant oxidation states.115 In this particular synthesis (Scheme 2-4), the catalytic cycle begins with an oxidative addition of a pre- activated Pd(0) species (60)‡‡‡ with the aryl halide (53) resulting in the formation of a Pd (II) complex (61).116
Scheme 2-4. Mizoroki–Heck cross-coupling catalytic cycle between 1,3,5- tribromobenze (53) and methyl 2-(4-vinylphenyl)acetate (54).
‡‡‡ In other instances, a Pd(II) precatlayst (i.e Pd(OAc)2) can be used which can be reduced in situ with a base. 2.1 Synthesis of 135C | 25
The subsequent species containing a new Pd-C σ-alkyl bond is very reactive towards carbon-carbon π-bonds115 such as the olefinic moity within styrene 54, and thus forms the complex 62. Frequently branded as the migratory alkene insertion step, the olefin of the alkene then adds across the carbon-palladium bond in a concerted manner and forms an organopalladium intermediate (63). During this process, the new carbon-carbon bond is formed;116 the electronic properties of this step also governs the overall regioselectivity of the reaction.
In this reaction process, the trans-configuration of the product is favoured due to the steric hindrance between the two phenyl groups of the styrene and the phenyl halide (for a more in-depth discussion of the regioselectivity of the Mizoroki–Heck reaction, see Section 7.2).116–118 At this point in the catalytic cycle, free rotation about the single C-C bond occurs until the palladium atom and one of the β-hydrogens are syn-coplanar (Scheme 2-5). This allows the intermediate (64) to undergo a key syn β-hydride elimination process to provide the desired product (65), as well as the essential palladium hydride species (66).
Scheme 2-5. Syn- (64) and Anti- (67) configurations of the analogues.
At the end of the catalytic cycle, the palladium hydride species (66) undergoes reductive elimination through reaction with a base (specifically in this case, N,N- dicyclohexylmethylamine), which regenerates the coordinately unsaturated Pd(0) species for its continuation in the catalytic cycle. For the synthesis of 135C, three consecutive catalytic cycles are required for the tribromobenzene substrate to cross- couple with three styrenes for the formation of the tri-styrenyl-substituted product (67). Conditions used for this reaction are based on Gregory Fu’s earlier work on Heck 119 reactions using the electron rich phosphine P(t-Bu)3. In this case, 5 mol% of 26 | Chapter 2: Synt hesis, Bioisosteres and Protein Interaction
§§§ Pd2(dba)3∙CHCl3 was used with [(t-Bu)3PH]BF4 in a 1:1 ratio, along with four equivalents of Cy2NMe in DMF; these conditions were able to provide 67 in a good 82 % yield (spectrometric data discussed in following section).
Conditions and reagents: Pd2(dba)3∙CHCl3 (5 mol%), [(t-Bu)3PH]BF4 (5 mol%), Cy2NMe (4 equiv.), DMF, 80 °C, 16 h; b) LiOH.H2O (9 equiv.), EtOH/H2O (1:9), 100 °C, 17 h.
Scheme 2-6. Mizoroki–Heck cross-coupling reaction between 1,3,5-tribromobenze (53) and methyl 2-(4-vinylphenyl)acetate (54), and the subsequent saponification of 67 to 135C (52).
In the final step of the synthesis of 135C, a simple saponification method was performed to remove each of the methyl ester moieties and to reveal the three carboxylic acids. When 67 was treated with nine equivalents of LiOH in ethanol/water (1:9) at reflux, the desired tricarboxylic acid product was furnished (52) in 57 % yield (Scheme 2-6). The major product isolated from this reaction contained no resonance at δ = 3.72 ppm in the 1H NMR spectrum which were observed in the starting material assigned to the methyl ester protons. Furthermore, the presence of a broad O-H peak at 3026 cm-1 by IR analysis and a distinctive molecular ion in the HR-MS corresponding to C36H31O6 confirmed the structure of this compound. An X-ray crystal structure of 52 was also obtained (detailed discussion in 2.1.1).
In the following section, spectrometric data of 67 and crystallographic data of 52 are discussed, representative of most analogues synthesised in this study; key spectrometric features of the successfully cross-coupled tristyryl core are established.
§§§ 476 For best results, the Pd2(dba)3∙CHCl3 complex was freshly prepared from a PdCl2 and dba. 2.1 Synthesis of 135C | 27
2.1.1 Discussion of the spectrometric data of 67 and crystallographic data of 52 Spectrometric data of compound 67
Figure 2-1. Structure of 135C precursor 67.
Due to the symmetrical nature of these tri-substituted motifs (Figure 2-1), the corresponding chemically equivalent hydrogen and carbon atoms of the three symmetrical units appear as a single resonance. Therefore, signals within the hydrogen and carbon spectra are relatively simple to assign and contain the following distinctive patterns in the aromatic region (Figure 2-2). In the 1H NMR spectrum of 67, a singlet at δ = 7.55 ppm can be assigned to the three aromatic protons of the central aromatic ring
(H2). The three aromatic rings attached to the central core contain AA’XX’ spin systems that present themselves as a first order A2X2 patterns, where JAX = JA’X’. Slightly upfield at δ = 7.52 are a set of apparent doublets (JAX = JA’X’ = 8.2 Hz) assigned to the six aromatic protons (H5’/H7’) closest to the carboxylic ester groups. The coupling constant is reflective of the ortho-coupling to the remaining set of aromatic protons (H4’/H8’) assigned as a doublet at δ = 7.30 (JAX = JA’X’ = 8.2 Hz). Two sets of doublets (J = 16.3
Hz) are assigned to the cross-coupled alkene at 7.19 ppm (H2’) and 7.13 ppm (H1’); the coupling constants are typical of vicinal trans-orientated protons. The upfield region shows two distinct singlets at δ = 3.72 ppm and δ = 3.66, which are assigned to the nine methyl ester protons (H11’) and six methylene protons (H9’), respectively. 28 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction
H2 H5’/H7’ H4’/H8’ H2’ H1’ H11’
* H CDCl3 9’
1 Figure 2-2. H NMR spectrum of compound 67 at 400 MHz in CDCl3. Solvent peak is marked with an asterisk.
In the 13C NMR spectrum of 67, a resonance at δ = 172.1 ppm was assigned to the carbonyl carbons of the carboxylic ester groups (C10’) (Figure 2-3). NMR spectrum simulations using the software ChemDraw® Ultra 12 were used to assist in the assignments of the aromatic quaternary carbons at δ = 138.2 (C3’), 136.3 (C1) and 133.6
(C6’). The two relatively higher resonances can be assigned to the 12 carbons in the outer aromatic rings; due to the proximity of these carbons to the carboxylic ester group, the outer carbons (C5’/C7’) are assigned to be at δ = 129.8 ppm and the inner carbons
(C4’/C8’) at δ = 126.9 ppm. Due to the same effect, the styrenyl carbons, C2’ and C1’, can be assigned to δ = 129 and 128.5 ppm, respectively. The remaining aromatic resonance at δ = 124.1 ppm is assigned to the tertiary carbon of the inner ring (C2). The aliphatic region of the spectrum displays two peaks at δ = 52.3 and 41.1 ppm which were unambiguously ascribed to the methyl (C11’) and methylene (C9’) carbons, respectively.
2.1 Synthesis of 135C | 29
C5’/C7’ C4’/C8’ * CDCl3 C1 C C C2’ 3’ 6’ C1’ C2
C11’ C9’
C10’
13 Figure 2-3. C NMR spectrum of compound 67 at 100 MHz in CDCl3. Solvent peak is marked with an asterisk.
The IR spectrum shows aromatic C-H stretches at 3026 and 2950 cm-1 and a strong band at 1730 cm-1 resulting from carbonyl stretching. As expected, multiple medium bands in the 1434 – 1585 cm-1 region are typical of aromatic C=C stretching. Sharp bands present between 1153 cm-1 and 1235 cm-1 represent in-plane C-H bond bending of the aromatic ring, whereas out-of-plane counterpart occurs in the fingerprint region around 794 cm-1.120
The ES+ high resolution mass spectrum of compound 67 shows a signal at m/z = 601.2590 with 100 % relative intensity which can be assigned to the molecular ion peak 79 81 C39H37O6. The absence of distinctive 1:1 Br and Br isotopic signals indicates the complete removal of bromine from the central aromatic core.
Crystallographic data of 52 (135C) The X-ray crystallographic structure of 135C could be determined from solvated crystals grown using vapour diffusion techniques in a THF/hexanes solvent system. From Figure 2-4a, it is clear that 135C has a trans-alkene configuration. Of the three styryl substituent groups, two are in the form of a bladed propeller about the central phenyl ring, whereas the third is rotated about the CPh -C bond. Interestingly, the data obtained resulting in Figure 2-4b illustrates the hydrogen bonding between two molecules of 135C. The two molecules form an unusual hydrogen bonded dimer where two carboxylic acid groups of one molecule are hydrogen bonded to two carboxylic acid 30 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction groups of the other molecule. The two pairs of hydrogen bonds presented very similar bond lengths (≈ 1.82 Å), but bond angles were varied between the pairs (161.8°/164.3° and 176.9°/175.1°). The third carboxylic acid group of each molecule form hydrogen bonds with the oxygen atoms of solvent THF molecules; the two hydrogen bonds have quite different bond lengths and angles, perhaps due to the different orientation of the THF molecules in relation to the carboxylic acid groups.
(a) (b)
Figure 2-4. (a) X-ray crystallographic structure of 135C onto the plane of the central phenyl ring; (b) The hydrogen bonded dimer of 135C projected along the a axis. Ellipsoids are drawn at the 50% probability level. Full crystallographic details are presented in the appendix A of this work.
2.2 Bioisosteric analogues of 135C The acidity of the carboxylic acid in a pharmacophore has been reported to be essential for retaining the antimicrobial activity of some compounds in the literature.100 Previous analogue studies on 135B showed that replacing the acid with other functional groups that do not contain this acidic proton result in a loss of activity in vivo, and hence inferred that that the carboxylic acid functional groups are the main pharmacophoric units of 135C. Unfortunately, biological assays of both 135B and 135C showed a decrease in antimicrobial activity when conducted in the presence of biological protein.94
In light of these observations, a literature search revealed, as suspected, that many drugs that contain benzoic acids bind to serum albumins, for example, salicylates and ibuprofen.35,45,121,122 As 60% of the total mass of plasma proteins is made up of serum albumins,123 in vivo activity of drugs with carboxylic acid moieties is expected to 2.2 Bioisosteric analogues of 135C | 31 decrease due to this effect; this adverse binding will lower the proportion of administered drug that is free to react in the cytoplasm.124,125 However, this is expected as many examples exist where carboxylic acids are known to be critically involved in the binding to targets specifically due to their charge-charge interactions.126
It is clear that there is a correlation between the pKa of the acid and the binding of the acid to HSA. Quantitative structure-activity relationships (QSAR) have shown that as 100,126– the pKa of the analogues increase, the affinity for protein binding also increases. 128 It has also been reported that substitution of the acid group with a range of different functional groups has negative effects on bioactivity, especially when the acidic moiety is the pharmacophoric unit of the drug.101 Hence, replacing the carboxylic acid moieties in 135C may be not be an immediately viable option. Previous work by Romero and co- workers indicated that if a balance is achieved between the reduction of the pKa values for minimal HSA binding, it may also retain the functionality at the ionisable proton for potent antimicrobial activity.100 Given this report, several bioisosteres were targeted through synthetic approaches to explore the activity and protein binding properties of different analogues of the lead compound 135C.
Isosterism is defined as “the similarity of molecules or ions which have the same number of atoms and valence electrons”.129 Isosteres, molecules that have similar chemical and physical properties,130 that produce similar biological activity are called bioisosteres.131 They are categorised into two types; classical and nonclassical. Classical bioisosteres are molecules that are similar in their steric and electronic properties, as well as the same count of atoms as the parent molecule.130,131 Nonclassical bioisosteres are molecules that imitate the spatial arrangement, electronic properties, or some other physicochemical properties of the parent molecule or substituent, allowing the retainment of biological activity.131
As compound 135C has excellent pharmacological activity but characteristics that limit its bioavailability, it seemed valid that replacing the carboxylic acid group with bioisosteric substituents that contain an ionisable proton with varying pKa values****
**** Using the SPARC programme,45 theoretical acid dissociation constants were calculated for 135C and the bioisosteric analogues.151 Each of the three analogues contain three ionisable protons; they are significantly far apart in space and electronically have little effect over each other, and hence have the same acid dissociation values for all three speciations (Table F2).
32 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction may be able to potentially overcome the protein binding problem, if the compounds retain a similar level of antimicrobial activity. In this work, we envisaged three bioisosteric replacements of carboxylic acid (Figure 2-5); sulfonic acid (68), sulfonamide (69) and tetrazole (70). As such, all were targeted through synthetic means.
Figure 2-5. The functional groups sulfonic acid (68), sulfonamide (69) and tetrazole (70).
2.2.1 Sulfonic acid analogue Sulfonic acids have nonplanar structures, and are far more polar and acidic than carboxylic acids, with pKa values of less than 1. There are many examples in the literature of carboxylic acid replacements with sulfonic acid that exhibit useful biological activities. Noteworthy examples include analogues of amino acids that are glutamate receptor activators,132 and derivatives of γ-aminobutyric acid (GABA) receptor antagonists133 and cholecystokinin-B (CCK-B) receptor antagonists.134
Attempted synthesis of the sulfonic acid analogue (71) was first carried out through a Mizoroki Heck cross-coupling reaction between commercially available compounds tribromobenzene 53 and the sodium salt 72 (Table 2-1). The previously discussed conditions for the synthesis of 135C (Table 2-1, entry 1) were used, along with two
Table F2 - Acid dissociation values of ionisable analogues.
Analogue 135C Tetrazole Sulfonic acid Sulfonamide
R =
pKa1/2/3 4.34 5.09 0.64 9.93
2.2 Bioisosteric analogues of 135C | 33
other literature conditions, using Pd(OAc)2 as the catalyst, for cross-coupling between aryl halides and styrenes;135,136 however, in all three cases, the desired product was not isolated.
Table 2-1. Attempted synthesis of 71 via the Heck cross-coupling reaction.
Entry Catalyst (mol%) Ligand (mol%) Base (equiv.) Sol. Temp. T (h) t 1 Pd2(dba)3CHCl3 (15) Bu3PBF4H (60) Cy2NMe (4) DMF 80 °C 19
2 Pd(OAc)2 (15) (o-Tol)3P (25) i-Pr2EtN (4) DMF 80 °C 40 3 Pd(OAc)2 (7.5) (o-Tol)3P (15) Et3N (7.5) DMF 100 °C 24
From preliminary TLC analysis, the reaction mixture showed the presence of a compound which was neither the starting material nor the coupling partner, as indicated by its fluorescence under long-wavelength UV light. This, along with the consumption of the limiting reagent, suggested the completion of the reaction under all three conditions trialled. Extraction of the reaction mixture with diethyl ether was unsuccessful and it was assumed that the product was still present in the aqueous layer. At first, the difficulty of the extraction was thought to be due to the presence of the analogue in its ionised form. However, even after acidifying the aqueous phase to a pH of less than 1, no compounds were isolated after extractions with a variety of different solvents. Although sulfonic acids have been shown to be able to survive and aid in Heck cross-coupling reactions as ligands,137 no examples where it is a functional group on the styrene coupling partner have been found in the current literature. 2.2.2 Sulfonamide analogue Sulfonamide was first used as a carboxylic acid substitute in protonsil (7)- related antimicrobial agents in the 1930s.138 In this example, the sulfonamide moiety shows similar hydrogen bond geometries to the carboxylic acids due to the similar distances 34 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction between the two oxygen atoms in both functional groups.138,139 In comparison to its carboxylic acid counterparts, sulfonamide derivatives are much less acidic (pKa ≈ 10), and have a nonplanar structure.
Figure 2-6. Structure of proposed sulfonamide analogue (73).
For the synthesis of the sulfonamide analogue 73 (Figure 2-6), vinylbenzenesulfonamide (74) was first prepared from sodium 4-vinylbenzenesulfonate (75) via a two-step literature preparation (Scheme 2-7).140,141
Scheme 2-7. Conversion of sodium 4-vinylbenzenesulfonate (75) to 4- vinylbenzenesulfonamide (74).
Sodium 4-vinylbenzenesulfonate (75) was first treated with thionyl chloride to give the intermediate, 4-vinylbenzene-1-sulfonyl chloride (76), which was immediately treated with ammonia. In the first attempt of this amination, aqueous ammonia was used as the reagent,140 providing a yield of 30 %. It was deduced that this low yield may have been due to the presence of water, effecting the amination process.115 In an attempt to increase the yield, anhydrous ammonia was used as an alternative.141 An excess amount of ammonia was condensed in the presence of the sulfonyl chloride compound, and a yield of 81 % of 74 was achieved. The success of the reaction was immediately confirmed when the 1H NMR spectrum of the isolated compound indicated a singlet resonance at 4.80 ppm assigned to the two sulfonamide NH2 protons. 2.2 Bioisosteric analogues of 135C | 35
Following the synthesis of the sulfonamide styrene substrate (74), Heck cross-coupling reactions were performed under the same conditions as above (Table 2-1) for the attempted synthesis of analogue (73) (Scheme 2-8).
Scheme 2-8. Attempted synthesis of 73 via the Heck cross-coupling reaction.
Preliminary TLC analysis indicated the completion of the reactions by the consumption of 53 and the presence of a new compound that fluoresces under long-wavelength light. Several extraction processes were carried out, but the product of the reaction was not isolated.
2.2.3 Tetrazole analogues Tetrazoles are one of the most frequently used isosteres of carboxylic acid in medicinal chemistry.142,143 In contrast to the other two bioisosteres, tetrazoles have similar planarity and acidity to that of corresponding carboxylic acids with pKa values of around 5,144,145 as well as being larger in space. The hydrogen bonding environment of the isostere is also deemed to be more extended than that of corresponding carboxylates,146 and its anion comparatively more lipophilic.147
It was envisioned that two tetrazole analogues (75 and 76) reflecting the structures of 135B and 135C could be accessed via their nitrile counterparts (Figure 2-7). A strategy was devised where the tetrazole functional group could be incorporated at an early stage prior to the Heck cross-coupling reaction. 36 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction
Figure 2-7. Structures of the proposed tetrazole analogues 75 and 76.
To this end, a nitrile functionalised phenyl aldehyde 77 was chosen as the precursor (Table 2-2). At first, 77 was treated with sodium azide and iodine in DMF at 120 oC.52 However, no product was isolated, possibly due to the decomposition of the starting material. A second reaction grinding 77 with zinc chloride and sodium azide before heating under solvent-free conditions at 90oC was trailled.148 Computational studies have shown that zinc(II) salts promote tetrazole formation by coordinating the nitrile to the zinc ion;149 this coordination lowers the energy barrier for the impending nucleophilic attack by the azide. The first attempt at this reaction was conducted under normal atmospheric conditions; as zinc chloride is hygroscopic, it was assumed that the grinding of the solids in air allowed the absorption of water into the mixture, which contributed to the low 20 % yield of tetrazole 78. To increase the yield of this reaction, the next attempt was conducted under a simple cone of nitrogen, which resulted in a slightly higher yield of 29 %. A third attempt was carried out, where the preparation of the starting materials was conducted in an argon-filled glove box. In this example, a fine grey powder was achieved but the reaction only furnished 78 in 23 % yield. For all three attempts, starting material 77 was also recovered from the reactions. As the three reaction conditions with different physical parameters resulted in similar yields, it was concluded that water does not have a dramatic effect on the cycloaddition. The identity of the product, 4-tetrazole benzaldehyde (78), was confirmed by a resonance at 160.8 ppm in the 13C NMR spectrum, assigned to the tetrazole carbon.150 Additionally in the 1H NMR spectra, there was a slight upfield shift of the aromatic proton resonances in comparison to those of the starting material.
2.2 Bioisosteric analogues of 135C | 37
Table 2-2. [2+3] cycloaddition of sodium azide and 4-cyanobenzaldehyde (77).
Entry NaN3 Catalyst/ Solvent Temp. Yield equivalents reagent o 1 1.5 I2 (cat.) DMF 120 C n.r. o 2 1.5 ZnCl2 (1.5 - 90 C 20 – 29 % equiv.)
Compound 78 was then subjected to a Wittig reaction (Scheme 2-9) using previously optimised reaction conditions;151 however, the desired reaction product 79 was not isolated. Interestingly, triphenylphosphine oxide was sequestered during the workup of the reaction mixture. The presence of this compound normally indicates the formation of the corresponding olefin. In our case, it was speculated that the product may be present in the aqueous layer of the extractions. This process was repeated, however, the desired product was not isolated in several extraction attempts.
Scheme 2-9. Wittig reaction of 4-(1H-tetrazol-5-yl)benzaldehyde (78).
As the solvent-free conditions described above resulted in varying in yields and limited attempts on further reactions of 77, another set of conditions by Koguru152 using sodium azide, triethylamine hydrochloride was applied to the nitrile styrene 80,†††† which gave the desired product 79 in a good 82 % yield (Scheme 2-10). Again, this success was indicated by the downfield shift of the aromatic signals in the carbon spectra, as well as
†††† Styrene 80 was accessed through the Wittig reaction of 77, as previously described.151 38 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction the physical change from liquid to a solid. These conditions were also applied to the nitrile 59 for the corresponding tetrazole 81 in 71 % yield.
Scheme 2-10. [2+3] cycloaddtion of NaN3 and nitriles (80, 59) for tetrazoles (79, 82).
These two tetrazole styrenes 79 and 81 were subjected to the Mizoroki–Heck cross- coupling reaction conditions (Pd2(dba)3∙CHCl3 (5 mol%), [(t-Bu)3PH]BF4 (5 mol%),
Cy2NMe (4 equiv.), DMF, 80 °C, 16 h) with 53 for the desired tetrazole analogues. Unfortunately, the desired products were not synthesised, as neither starting materials were consumed in the reaction.
A second strategy was devised where the [2+3] dipolar-cycloaddition can be applied to the larger coupled scaffolds. 82 was previously prepared as an analogue of 135B, beginning with the Heck cross-coupling reaction between 80 and 1,3,5- tribromobenze.151 For nitrile 82, styrene 80 was coupled with 1,3,5-tribromobenzene
(53) (Pd2(dba)3∙CHCl3 (5 mol%), [(t-Bu)3PH]BF4 (5 mol%), Cy2NMe (4 equiv.), DMF, 80 °C, 16 h) at 39 % yield. Its success was confirmed by the downfield shift of the terminal alkene to δ = 7.20 – 7.06 ppm, as well as from the absence of one of the two geminal proton s at δ = 5.14 ppm.
An initial attempt at synthesising tetrazole 75 between sodium azide and nitrile 82 under the previously described solvent-free conditions with ZnCl2 was unsuccessful (Table 2- 153 3, entry 1). Another zinc(II) salt condition was trialled with ZnBr2 in H2O/n-butanol (Table 2-3, entry 2), however, decomposition of nitrile 82 was observed and no desired product was acquired.
Following, the Koguro conditions152 that successfully transformed the styrenes (Figure 2-10) were trialled with 82 (Table 2-3, entry 3). The first attempt using was 2.2 Bioisosteric analogues of 135C | 39 unsuccessful and only starting material was isolated at the end of the reaction. This is most likely due to the low solubility of 82 in toluene. Subsequently, the solubility of 82 was tested to find a more suitable solvent for the reaction. It was found that 82 was not soluble in THF and DMF, and slightly soluble in dioxane, however, the Koguru conditions were still unsuccessful following this change in solvent (Table 2-3, entry 4).
In a last attempt, the procedure as patented by Koo et al.154 was trialled with a change in physical conditions; the reaction was performed with 35 equivalents of both NaN3 and
NH4Cl and in a sealed tube environment. Under these harsh reaction conditions, 82 was successfully transformed into 75 in 92 % yield (Table 2-3, entry 5). This was confirmed by the presence of the tetrazole proton signal at 16.8 ppm. The nitrile 83, bearing an additional methylene, was also subjected to these conditions and was successfully transformed in 76 in 73 % yield (Table 2-3, entry 6). This was confirmed by the presence of the tetrazole proton signal at 16.2 ppm.
Table 2-3. [2+3] Cycloaddition of sodium azide and nitriles 82 and 83.
Entry NaN3 Reagents (equiv.) Solvent Temperatur Yield equivalents e
1 4.5 ZnCl2 (4.5) solvent-free 90 °C n.r. o 2 4.5 ZnBr2 (3.3) H2O/ n-butanol 100 C n.r.
3 3 NEt3.HCl (3) toluene 100 °C n.r. 4 3 NEt3.HCl (3) dioxane 80 °C n.r. o 5 35 NH4Cl (35) DMF 125 C 92 % o 6* 35 NH4Cl (35) DMF 125 C 73 % *n = 1.
40 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction
2.2.4 Miscellaneous analogues During the process of preparing bioisoteres of the 135C key compound, other analogues were envisioned to be synthesised either from transforming existing analogues or by changing the centre structure of 135C. In this process amide analogues 84 and 85 (Figure 2-8) were envisioned to be accessed via the nitrile analogues 82 and 83, respectively.
Figure 2-8. Structures of the proposed amide analogues.
The two nitriles were subjected to an acid-catalysed conversion into the amide by 155 stirring with trifluoroacetic acid-H2SO4 (4:1, v/v) at 75 °C. Consumption of the starting material was shown by the change of physical property of the solid collected at the end of the reaction. Unfortunately, the resulting compounds were insoluble in DMSO, as shown by the lack of any signals in the NMR spectra. A Solid 1C NMR analysis was conducted and the resulting spectra indicated signals at 129.26 ppm and 129.39 ppm, respectively, corresponding to the amide carbonyl carbons. Further, the neat IR spectra of both compounds showed no signals in the 2000-2500 cm-1 region, indicating that it is not the nitrile starting material; carbonyl (1605.85 and 1656.19) and amine (3188.19, 3188.73) signals were also detected.
2.2.5 Remarks on analogue syntheses It appeared that highly polar and fluorescent compounds were synthesised in the reactions for the sulfonic acid and sulfonamide analogues, but difficulties in the isolation of these compounds were encountered. Due to their highly polar nature, it would be plausible to tackle this problem by utilising an ion exchange resin or reverse- phase chromatography to isolate these products. In the scope of this thesis, the isolation of these products was not pursued further, as impracticality and time limitations 2.3 Interaction with serum albumin | 41 outweighed the importance of their isolation. As the tetrazole bioisoteres were successfully synthesised, it was sensible to pursue the investigation of the protein interaction problem with these analogues.
The synthesised tetrazole and amide analogues are expected to be problematic in antimicrobial assays due to their varying solubilities in DMSO. However, attempts at determining the MIC of the prior pair was carried out and described in Section 3.2.6.
The bioisosteric analogues were originally proposed to address the issue of decrease in antimicrobial activity when assays are performed in the presence of blood or protein. It was therefore sensible to establish that there was a definite interaction between 135C and protein.
2.3 Interaction with serum albumin Serum albumin is the most abundant plasma protein; it is also the major binding protein for acidic drugs,156–158 and a known transporter.159 Therefore, plasma protein binding displacement interactions affect the concentration of drug in the plasma that is free to interact with their target. Previously, we had shown that the antimicrobial activity of 135C was reduced in the presence 5% equine serum; the MIC against S. aureus NCTC 6571 was 2 μg/mL compared to 0.125 μg/mL in the absence of serum. A similar degree of decrease in activity of 135B also occurs. As the two structurally analogous compounds are both acidic, it was hypothesised that interaction with serum albumin was a likely cause for the observed decrease in in vitro antimicrobial activity.
The domain structure among all mammalian serum albumins is highly conserved,160 therefore the use of bovine (BSA), equine (ESA), and human (HSA) serum albumins in in vitro assays was highly comparable. To confirm the above-mentioned hypothesis, broth microdilution tests were conducted to determine MIC of 135C against S. aureus NCTC 6571 in the presence of BSA, a readily available lyophilised protein. With a final concentration of 4 μM BSA in each well, the MIC was determined to be 2 μg/mL. Protein crystallography and NMR spectroscopy methods were trialled to elucidate the interaction between BSA and 135C.
2.3.1 Protein crystal structure (X-ray crystal diffraction data collected by Dr. Gavin Knott, Dr. Amanda Lewis and Dr. Jason Schmidberger) 42 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction
X-ray crystallography is one of two methods which offer atomic resolution of 3D protein structures.161 It was considered that if BSA and 135C could be co-crystallised together, the X-ray diffraction data would shed light on the relationship between the protein and the drug, as well as providing quantitative analysis such as the number of drug molecules taken up by each individual protein.
Prior to the crystal growth process, the lyophilised BSA was treated with activated charcoal under acidic conditions to remove any fatty acids present.162 The sample was then purified using gel-chromatography. The vapour-diffusion hanging-drop method was used for crystallisation of the protein. Initially, BSA native crystals were obtained using PEG MME 5K and calcium acetate in MES buffer. These crystals were then soaked with a 200 mM ethanolic solution of 135C for one week. X-ray diffraction data was collected on the soaked crystals, and solved. However, the crystal structure solved (2.3 Å) was that of native BSA with no 135C in the structure.
An alternate method was attempted where the complex of BSA and 135C was formed by mixing the defatted and purified albumin at 1 mM concentration in buffer (100 mM NaCl and 10mM Tris at pH 7.4) with 10-molar excess of 135C. The mixture was incubated overnight at 35 °C with stirring, and centrifuged before crystallisation setup. In this process, it was evident that 135C was taken up by BSA, as the originally clear and colourless solution became a clear and yellow solution after incubation (Figure 2-9). This protein/drug solution was subjected to the crystallisation setup conditions as previously used; unfortunately, no crystals were obtained from this experiment.
I II Figure 2-9. Solution of BSA before (I) and after (II) incubation with 135C.
2.3 Interaction with serum albumin | 43
2.3.2 NMR studies of 135C/BSA interaction (NMR experiments performed and processed by Dr. Mark J. Howard) Saturated-transfer difference (STD) NMR‡‡‡‡ is a versatile and convenient method that allows for the observation of the interaction between small molecules and protein targets (“ligand observe”). It is the ‘gold standard’ technique that is often utilised in NMR-based fragment-based drug discovery,163 and has many important applications in biological systems.164 The method has been used extensively for screening, identifying and characterising ligand binding to receptors such as free proteins,165–167 lipids,168,169 cells,170,171 and viruses.172,173
Two NMR samples were prepared with 1mM of 135C in (~7%) DMSO-d6, PBS buffer at pH 6.8 and D2O; one sample contained 20 µM of BSA (experimental sample), and the other no protein (negative control). STD NMR spectra are as follows (Figure 2-10).
STD NMR (- BSA)
STD NMR (+ BSA)
HDO* *DMSO 135C
135C
1 BSA H NMR
Figure 2-10. 1H NMR of 135C/BSA and STD NMR spectra of negative control (- BSA) and experimental sample (+ BSA).
‡‡‡‡ An STD NMR is the difference experiment between a saturation transfer spectrum and a regular NMR spectrum to observe a ligand that binding to a protein in the mM-nM dissociation constant range.8, When the protons of the target protein in a known characteristic region are selectively saturated, NOE and spin diffusion will spread the magnetisation across the entire protein. If a ligand binds during selective protein saturation, it can be transferred from protein to ligand. Such transfer attenuates the ligand 1H NMR magnetization that can be subsequently observed via a difference spectrum. The difference spectrum will only contain ligand signals when it binds and dissociates and confirms an interaction between the protein and ligand. 163,165,477 44 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction
From the STD (+BSA) spectrum (blue), it can be seen that there is a definite interaction between the protein and 135C, as confirmed by the presence of its signals. Unexpectedly, a resonance at δ = 1.89 ppm is also present in the BSA characteristic region. This signal is also present in the negative control spectrum (yellow) and does not correspond to any atoms in the 135C structure. As there is no BSA present in this sample, this signal was treated as an unrelated NMR artefact, which was confirmed in the complementary Water-Ligand Observed by Gradient SpectroscopY (WaterLOGSY) experiment.
A related saturation transfer approach to ligand-observe binding to proteins is the WaterLOGSY experiment that transfers magnetisation through NOE and spin diffusion via bulk water molecules (and consequently bound ligands) in the sample.163 This technique can distinguish between water molecules that are bound as a complex (such as water molecules associated with protein), and the water molecules free in solution; the latter signals appear inverted in the spectra due to a change in sign of the NOE.174 WaterLOGSY is also used across NMR-based pharmaceutical drug screening because it is complimentary and assists in quality control and identification of false-positive STD NMR peaks.175–177
The BSA-positive WaterLOGSY spectrum (Figure 2-11, blue) shows signals associated with 135C (resonances in the aromatic region and at δ = 3.54 ppm) and other atoms (resonances at δ = 6.73 ppm and between δ = 0.95 – 2.16 ppm). These upright resonances indicate that transfer has occurred between protons from ‘ordered water molecules’ with those in the sample. Essentially, these transfers are between protons within bound water-complexes.
The BSA-free WaterLOGSY spectra (Figure 2-11, red) shows inverted 135C signals, along with other upright resonances at δ = 6.73 ppm and between δ = 0.95 – 2.16 ppm. This spectra shows that 135C is associated with water molecules that are free in solution, and that the other resonances are not part of the 135C molecule, confirming them as artefacts. As there is no protein present in this sample, it is implied that these artefact presented upright resonances due to self-association that is not related to the presence of 135C. This confirms these signals as artefacts within the STD NMR 2.3 Interaction with serum albumin | 45 spectra from Figure X and not associated with the interaction of 135C with BSA. The species creating these signals are most likely to be impurities within the sample.
135C 135C WaterLOGSY (+ BSA)
Self- association artefacts from impurities WaterLOGSY (- BSA)
HDO* *DMSO
Figure 2-11. WaterLOGSY NMR spectra of negative control (- BSA) and experimental sample (+ BSA).
Despite excellent agreement between STD NMR and WaterLOGSY, a third supplementary ligand-observe experiment was conducted utilising the Carr-Purcell- Meiboom-Gill (CPMG) pulse sequence method, to provide irrefutable evidence for 135C interaction with BSA.§§§§ CPMG experiments detect ligand binding by registering an increase in ligand NMR line widths in the presence of protein.163 This increase in line width caused by an increase in apparent molecular weight of the bound-ligand species shortens the spin-spin T2 relaxation time of the nuclei in question and broadens the line.163,178 When a protein interacts with atoms within a ligand, the corresponding resonances will experience a line-broadening effect, which is seen in the spectrum as a decrease in signal intensity in the presence of protein.163 Therefore, in order to observe binding by CPMG, it is crucial to perform this NMR experiment identically with the same concentration of ligand in the presence and absence of protein.
The CPMG experiment produced near identical spectra (Figure 2-12) from the BSA and BSA-free samples, apart from a decrease in signal intensity at δ = 3.54 ppm (marked by asterisk). Since the signal intensity is only decrease and not eliminated, the binding event is construed to be weak. This change in the spectra indicated that the added
§§§§ CPMG allows for the measurement of spin-spin T2 relaxation times of any nuclei. 46 | Chapter 2: Synthesis, Bioisosteres and Protein Interaction protein interacts with the methylene protons of 135C, and not with the aromatic core of the structure. From this result, it may be inferred that BSA binds to the extremities of the 135C structure, endorsing the hypothesis that serum albumin does indeed bind at the carboxylic acid moieties.35,45 Weak binding is confirmed through all three ligand- observe NMR experiments. Saturation transfer techniques require ‘weak’ binding to produce a positive result; the ligand must bind to become saturated and dissociate to be detected in both STD NMR and WaterLOGSY experiments. These experiments observe ‘weak’ binding in the mM-nM range with the greatest sensitivity being in the μM dissociation constant regime.166
It is noted that apart from the artefact peaks as seen previously, a signal at δ = 3.76 ppm is present. This resonance may be due to the degradation of the 135C structure in the presence of DMSO over time.
* CMPG (+ BSA)
CMPG * (- BSA)
Figure 2-12. CPMG NMR spectra of negative control (- BSA) and experimental sample (+ BSA).
From observations made from these three NMR methods, it was confirmed that BSA interacts with the 135C structure. The STD and WaterLOGSY techniques showed that magnetisation is transferred through NOE and spin diffusion to the bound ligand via the protein, and protein-bound water molecules, respectively. The CPMG experiment suggests that 135C is bound to BSA closest to its methylene protons, alluding to a binding site at the carboxylic acid moiety, which is supported by observations in the literature.35,45
2.3 Interaction with serum albumin | 47
48 | Chapter 3: Biologic al Evaluations of 135C
Chapter 3: Biological Evaluations of 135C 3.1 Brief introduction The bioactivity of an antimicrobial agent is important in many ways. Its spectrum of activity against an array of microorganisms can highlight its application in the real world: what can I use this drug to treat? Its cytotoxic properties give us a better understanding of its chemotherapeutic potential or potential adverse effects. Resistance studies on antimicrobial compounds are also very important in the current climate; will this drug be a viable option to treat infections efficiently? Or will it contribute to the antibiotic resistance problem that the health sector is currently facing? The following chapter investigates the above-mentioned properties and examines how these data affect the viability of this compound.
3.2 In vitro susceptibility tests When a medical practitioner prescribes an antimicrobial agent for treating an infection, they need to determine the dosage amount based on a number of factors; the individual’s weight and height, the pharmacokinetic and pharmacodynamic properties of the chosen drug, as well as the degree of bacteriostatic or bactericidal activity of the drug. Hence, in vitro susceptibility is an important characteristic to explore when evaluating a new antimicrobial compound; the minimum inhibitory concentration (MIC)***** and minimum bactericidal concentration (MBC)††††† are vital to support the clinical relevance of the agent.
3.2.1 Spectrum of activity Antimicrobials are proficient at inhibiting the growth of specific ranges of microorganisms.179 The susceptibility of a bacterium to an antimicrobial is what determines whether the drug is of any use in the treatment of particular infections, and it was therefore important to determine the spectrum of antimicrobial activity of 135C. These ranges of activity can be classified as narrow or broad, and each antimicrobial type has its own advantages and disadvantages.
Narrow-spectrum antimicrobial compounds affect only a limited number of microorganisms, whereas an antimicrobial that has broad-spectrum activity may inhibit a range of Gram-positive and negative microorganisms. An advantage of using a broad-
***** The lowest concentration of drug capable of preventing microbial growth ††††† The lowest concentration of drug that is needed to kill the microbe 3.2 In vitro susceptibility tests | 49 spectrum antimicrobial is that medical practitioners do not have to identify the specific bacteria causing the infection before prescribing the treatment. However, there are risks associated with broad-spectrum antimicrobials, for example, they are likely to also kill normal gut flora, which may lead to other problems.
3.2.2 Broth microdilution assay The broth microdilution test is a quantitative method that can be used to determine the MIC of an antimicrobial agent against a bacterial isolate.180 This method is one of the first antimicrobial susceptibility testing techniques developed and was originally performed in volumes of bacteriological growth media of 1 – 10 mL (macrodilution).181 Today, the procedure is carried out in microtiter plates (generally 96-wells), which allows the concurrent testing of multiple antimicrobial agents in a range of eight 2-fold dilutions (microdilution).181 In terms of drug discovery, this method has been reported to be used to test the activity of plant flavonoids against MRSA and VRE.182 Advantages of this method include the generation of accurate MICs, its easily reproducible nature, convenience of using prepared 96-well panels, as well as the low costs of reagents required.
In this assay, serial 2-fold dilutions of antimicrobial agents are prepared in the wells of a microtiter plate; this is followed by adding a standardised volume of a standardised bacterial suspension to the wells, and the plates are typically incubated at an appropriate temperature overnight.180 The MIC can then be determined as the lowest concentration of the antimicrobial agent that inhibited visible growth of the bacteria. To determine the MBC, a subculture can be taken from the MIC tests and inoculated onto agar plates in the absence of the antimicrobial compound. The following section describes a series of assays on clinically relevant pathogens to explore the spectrum of activity of 135C.
3.2.3 Susceptibility of Gram-positive bacteria The MIC and MBC for a series of pathogenic aerobic Gram-positive bacteria was determined. This included isolates of 20 Staphylococcus aureus, 10 Micrococcus sp., 10 Streptococcus pyogenes, 10 Streptococcus pneumoniae, and 20 coagulase-negative staphylococci. Susceptibility tests against this range of bacteria showed that 135C was most active against S. aureus, with MICs ranging from 0.12–0.5 μg/mL. The MBCs for all species tested were ≥ 32 μg/mL (Table 3-1); further determination of most values beyond 32 μg/mL was not conducted as they are clinically irrelevant for this subset of 50 | Chapter 3: Biological Evaluations of 135C bacteria. Given the large difference between the MIC and MBCs, the activity of 135C was deemed bacteriostatic.
3.2 In vitro susceptibility tests | 51
3.2.4 Susceptibility of anaerobic bacteria Susceptibility tests against a range of anaerobic bacteria showed that compound 135C was moderately active against the Gram-positive anaerobe Clostridium difficile with an MIC of 16 μg/mL, but less so towards Gram-negative anaerobes (Bacteroides spp. and Prevotella bivia) with a MIC range of 16 – 256 μg/mL (Table 3-2).
Table 3-2. MICs of 135C against Gram-positive and negative anaerobic bacteria (µg/mL). Organism MIC (µg/mL) Bacteroides fragilis NCTC 9343 256 Bacteroides distasonis ATCC 8503 16 Bacteroides fragilis NCTC 8560 32 Bacteroides thetaiotaomicron M760 a 256 Bacteroides vulgatus NCTC 10503 256 Clostridium difficile NSW132 a 16 Prevotella bivia IC7620 a 32 a clinical species
As S. aureus can grow in both low-oxygen and completely anaerobic conditions,183 a broth microdilution assay was conducted to investigate the effect of oxygen conditions on the activity of 135C. When MICs were determined under strict anaerobic conditions against S. aureus NCTC 6571, the value decreased from 0.5 to 0.004 μg/mL (7-fold change), suggesting that either the compound may be oxidised to a less active derivative under aerobic conditions, or that it may be metabolised differently. When S. aureus is grown under anaerobic conditions, ATP is synthesised by either fermentation or nitrate respiration instead of aerobic respiration of oxygen;184 therefore, another plausible hypothesis is that 135C interrupts one of these processes.
3.2.5 Susceptibility of Gram-negative bacteria The disk diffusion method is a semi-qualitative technique that is used to determine whether a bacterial isolate is susceptible, intermediate or resistant to a compound.180 The preliminary susceptibility testing of a number of Gram-negative bacteria with 135C (10 mg/mL) was performed using this method and all organisms showed no susceptibility except for M. catarrhalis NCTC 3625.94 This strain gave a zone of inhibition of 20 mm; a broth microdilution assay was also performed, and an MIC of 52 | Chapter 3: Biological Evaluations of 135C
0.25 µg/mL and an MBC of 2 µg/mL was determined. Further assays with another 10 strains of M. catarrhalis showed good activity with MICs ranging from 1 – 16 µg/mL and MBCs of ≥ 32 μg/mL.
3.2.5.1 Susceptibility of Gram-negative bacteria in the presence of PMBN The above-mentioned work showed that 135C does not have any appreciable activity against most Gram-negative bacteria; this low activity may be due to several mechanisms specific to Gram-negative bacteria such as reduced outer membrane permeability and active efflux systems. Previous studies have shown that polymyxin B nonapeptide (PMBN) interacts with the lipopolysaccharide of the outer membrane of Gram-negative bacteria.185 PMBN has been used to sensitise many bacteria to a range of hydrophobic antibiotics such as novobiocin, erythromycin and clindamycin.186 The compound PMBN alone does not cause leakage of periplasmic proteins from bacteria but will allow entry of hydrophobic antimicrobial compounds into the cell without causing any damage to it, facilitating hydrophobic diffusion through the outer membrane.187
In the current study, susceptibility tests against several strains of Escherichia coli and Pseudomonas aeruginosa showed that the activity of 135C was enhanced when tested in the presence of PMBN (Table 3-3), indicating that the outer membrane limits entry of 135C into the cell. The enhanced activity was more pronounced with E. coli than P. aeruginosa with the former exhibiting at least a 3-fold increase in susceptibility when PMBN was present. Furthermore, the susceptibility of M. catarrhalis to 135C is likely due to the lack of long O antigen polysaccharide chains in the lipopolysaccharide structure of its outer membrane, which are characteristic of Gram-negative enteric bacteria such as E. coli. The absence of these lipooligosaccharide (LOS) chains results in the increased permeability of the bacterial outer membrane,188 which likely contributes to the susceptibility of this organism to 135C.
3.2 In vitro susceptibility tests | 53
Table 3-3. MICs for Gram-negative bacteria in the presence and absence of PMBN (µg/mL). Organism MIC (µg/mL) 135C 135C Novobiocin Novobiocin + PMBNa +PMBNa E. coli ATCC 43889 >512 128 64 2 E. coli NCTC 10538 >512 32 256 4 E. coli ATCC 25922 >512 128 128 2 P. aeruginosa NCTC 6749 512 256 >512 n.d. P. aeruginosa ATCC 27853 512 n.d. 256 1 P. aeruginosa ATCC 25668 512 512 512 8 a 5.0 μg/mL n.d.= no data
3.2.6 Antimicrobial activity of analogues Two nitrile, two tetrazole and two amide analogues of 135C were synthesised as described in Section 2.2. These were synthesised in the hope that they would have antimicrobial properties but not have these properties decreased in the presence of serum albumin.
The two nitrile (82 and 83) and two tetrazole (75 and 76) analogues were tested against S. aureus NCTC 6571. Unfortunately, the two nitrile compounds showed no activity at concentrations up to 32 μg/mL. The MIC values of tetrazole analogues 75 and 76 against S. aureus 6571 were 0.5 and >32 μg/mL, respectively. When the experiments were conducted with 5% HAS added, both compounds showed MICs of >32 μg/mL. It is important to note that the solubility of the tetrazole derivatives was low, hence the MIC values may not be a true reflection of their antimicrobial properties. Due to their insoluble nature, the amide analogues (84 and 85) were not tested for their biological activity and deemed unviable in the pursuit of new antimicrobial agents.
Originally, synthesis of the bioisostere analogues was proposed as a method to tackle the decrease in activity in the presence of protein, whilst maintaining a similar biological activity to the parent 135C. Although tetrazole 75 retained a similar MIC of 0.5 μg/mL, its antimicrobial activity was decreased when HSA was present in the assay. Even though the tetrazole is larger in size than carboxylic acid, this result suggests that 54 | Chapter 3: Biological Evaluations of 135C the tetrazole moiety interacts with serum albumin. This interaction has also been reported in the literature.189,190
3.3 Toxicology studies To be clinically useful, an antimicrobial agent needs to have an acceptable toxicity profile. It is critical that cytotoxicity of a compound be measured in parallel with its pharmacological activities.191 Briefly, cytotoxicity is the capability of a compound or mediator cell to induce living cells to undergo necrosis or apoptosis. Cytotoxic assessments in the development stage of new pharmaceuticals are vital to confirm the safety of the end-users. To predict whether a compound will have potential toxic effects in vivo, a range of tests on the viability of both prokaryotic and eukaryotic cells can be conducted.192 The following outlines two in vitro toxicology studies; an assay of haemolytic activity and the Ames Salmonella/microsome mutagenicity assay.
3.3.1 The Ames Salmonella/mutagenicity tests The Ames mutagenicity test is an assay which utilises bacteria to determine whether a compound can cause a change in a DNA sequence. Specifically, the assay uses mutated Salmonella typhimurium strains as an indicator to observe whether a chemical has mutagenic activity. The main mutation of these Salmonella strains renders them unable to synthesise histidine, however, if a compound is mutagenic, it can revert the mutation back and the bacteria will then be able to synthesise histidine and grow in the absence of exogenous histidine.
3.3.1.1 Mutagenic strains In this investigation, we chose three commonly used mutated Salmonella strains: TA98, TA100, and TA1535. All three strains have rfa and uvrB mutations: the rfa mutation leads to a weak lipopolysaccharide layer which increases the permeability to larger molecules, and the uvrB mutation eliminates excision repair of DNA damage.193 Both the TA100 and TA1535 strains contain the same histidine mutation hisG46, a missense (base pair substitution) mutation.194 It is an AT GC mutation that leads to a change in the first enzyme in the histidine biosynthetic pathway, ATP-phosphoribosyltransferase. This base pair substitution occurs in the polypeptide chain where a proline (codon CCC) is inserted instead of a leucine (CTC).195 These two strains are used to detect mutations that revert bacteria to their wild-type state, which is triggered by mutagens that cause GC base-pair substitutions. The difference between the TA1535 and TA100 is an 3.3 Toxicology studies | 55 addition of a pKM101 plasmid in the latter; this plasmid contains the mucAB genes whose products enhance SOS mutagenesis (error-prone repair of DNA damage).195 The addition of this plasmid makes TA100 the most sensitive of all of the S. typhimurium tester strains.196 The TA98 strain has a frameshift mutation, and similar to TA100, it also contains the pKM101 plasmid. This strain is used to detect mutations caused by frameshift mutagens.
3.3.1.2 Mutagenic activity of 135C A ‘positive’ result in this test is highly indicative of rodent carcinogenicity as it shows that the chemical used has reverted the mutated Salmonella strains back to its wild-type state. The results from the Ames mutagenicity tests of 135C against TA98, TA100 and TA1535 are as outlined in Table 3-4. 135C gave a ‘negative’ result as the number of colonies on agar plates containing the compound was not greater than on the negative control plates. Results of this mutagencity test suggests that 135C does not have mutagenic properties.
Table 3-4. Mutagenicity of compound 135C determined by the Ames test with three Salmonella strains Strain Control Compound (µg/plate) Negative Positive 100 300 1000 TA98 19 1293 23 19 12 TA100 120 1119 126 103 97 TA1535 27 2037 23 23 25 *Values represent mean numbers of colonies per agar plate
3.3.2 Haemolytic activity of 135C To determine its cytotoxic nature and as an indicator of eukaryotic cell membrane damage and cytotoxicity, 135C was tested for its ability to lyse sheep erythrocytes. The haemolytic activity of 135C was very low at concentrations up to and including 50 μg/mL but 13.01% of erythrocytes were lysed at 500 μg/mL (Table 3-5).197 No haemolysis was observed for the equivalent tests with DMSO (solvent control). 56 | Chapter 3: Biological Evaluations of 135C
Table 3-5. Haemolytic activity of compound 135C Concentration of 135C (µg/mL) Average (SD) % haemolysis (n=2) 0 0.0 ± 0 0.5 0.14 ± 0.5 5 -0.30 ± 0.19 50 0.03 ± 0.19 500 13.01 ± 1.67 PBS (positive control) 0.0 ± 0 SDW (negative control) 98.69 ± 0.02
3.4 Antimicrobial resistance studies As previously discussed in the introductory section, antimicrobial resistance is one of the most pressing problems in the health sector. Therefore, for an antimicrobial agent to be successful in the development pipeline, it is important that the target organism does not develop resistance quickly. The following experiments were conducted to investigate the capacity for organisms to develop resistance to 135C.
3.4.1 Serial passage of S. aureus isolates with compound 135C Four S. aureus strains were serially passaged with increasing concentrations of compound 135C over a period of 10 days to investigate the development of resistance (Figure 3-1).
Unfortunately, the experiments showed that after just the first passage, the MIC values were increased by at least 3-fold. The rate of development of resistance to compound 135C was rapid, as shown in Fig 3-1. It is interesting to note that the resistance was fairly stable after the initial dramatic increase. Serial passages of S. aureus isolates with compound 135C were performed over a period of 10 days, and the results showed a dramatic increase in MIC from initial values of 0.12-0.25 μg/mL to 32-64 μg/mL.
3.4 Antimicrobial resistance studies | 57
*clinical isolates
Figure 3-1. Serial passage of S. aureus strains with 135C.
3.4.2 Stability of resistance To establish whether the resistance was a temporary adaptation or a genetic mutation, MICs were determined again after 10 drug-free passages; the results were as outlined in Table 3-6. For all four strains tested, the MIC values after 10 drug-free passages were 32 μg/mL, similar to the values (32-64 μg/mL) determined on day 10 of the serial passage assay with compound 135C. This indicates that changes in susceptibility were stable, and were possibly due to a stable genetic mutation.
Table 3-6. Resistance study MIC results. Strain Time of MIC 135C MIC (μg/mL) S. aureus NCTC 10442 Initial 0.25 Final (10 passages) 32 After 10 drug-free passages 32 S. aureus ATCC 29213 Initial 0.25 Final (10 passages) 64 After 10 drug-free passages 32 S. aureus 15913 Initial 0.25 Final (10 passages) 64 After 10 drug-free passages 32 S. aureus 3784546E Initial 0.5 Final (10 passages) 64 After 10 drug-free passages 32
58 | Chapter 3: Biological E valuations of 135C
3.4.3 Cross-resistance studies of generated mutants The susceptibility of serially passaged S. aureus strains to a range of commercially available antibiotics was determined. The antimicrobials were selected to cover the five main targets for antimicrobials within bacterial cells. Cross-resistance to an antimicrobial could indicate a shared, or at least similar, mechanism of antimicrobial action.
The results generated in this study (Table 3-7) did not show any cross-resistance and, as such, did not reveal any new insights into the potential mechanism of action of compound 135C, as wild-type (parent) and serially passaged strains did not differ in susceptibility.
Table 3-7. Cross-resistance study with S. aureus wildtype strains, and isolates passaged with 135C. MICs (µg/mL). S. aureus strain CIP ERY GEN OXA RIF VAN KAN CHL 135C 10442 P 0.5 1 1 > 16 0.0078 1 4 8 > 8 10442 W 1 1 2 > 16 0.0078 1 8 8 4 15913 P > 8 > 16 > 16 > 16 0.0039 2 > 128 8 > 8 15913 W > 8 > 16 > 16 > 16 0.0078 1 > 128 16 0.25 3784546E P > 8 > 16 2 > 16 0.0078 1 4 16 > 8 3784546E W > 8 > 16 2 > 16 0.0078 1 8 16 0.5 29213 P 0.5 1 1 0.5 0.0078 1 4 16 > 8 29213 W 1 2 0.5 0.25 0.15 1 8 16 0.5 CHL, chloramphenicol; CIP, ciprofloxacin; ERY, erythromycin; GEN, gentamicin; KAN, kanamycin; OXA, oxacillin; RIF, rifampin; VAN, vancomycin; P = passaged with 135C; W = wildtype.
3.4.4 Growth fitness of generated mutants Often, the acquisition of bacterial resistance is accompanied by a fitness cost to bacteria,198 typically evident as a reduction in growth rate or cell density. To determine whether the isolates resistant to compound 135C had altered growth capabilities, fitness experiments were conducted to compare the rate of growth of the wildtype and the serially-passaged S. aureus isolates. The results are shown in Figure 3-2. Data from the current study indicate minor differences in the fitness of parent and serially passaged strains, with the greatest difference seen for S. aureus ATCC 29213.
The growth fitness of the serially-passaged isolates was somewhat less robust than the wildtypes for all four strains tested. For the first 7 h of growth, the rate at which the serially-passaged isolates grew was consistently slower than that of the wild type, 3.4 Antimicrobial resistance studies | 59 however, at t = 24 h, the OD600 of all the strains tested were comparable. This result is supported by several studies which demonstrate that there is a fitness cost associated with antimicrobial resistance.198 However, since the difference in growth rate was not very large, the fitness cost is likely to be insignifitcant; literature precedent shows that this characteristic can lead to mutations that are more likely to persist in the absence of antibiotic treatment.199
60 | Chapter 3: Biological Evaluations of 135C
3.4 Antimicrobial resistance studies | 61
3.4.5 Genomic comparison of generated mutants and wildtypes (genome mapping and analysis performed by PhD candidate Daniel R. Knight)
Acquired resistance results from genetic alterations, resulting in changes to an organism’s normal cellular physiology and structure.28 In some species such as Mycobacterium tuberculosis, genetic mutation is the sole cause of its clinical resistance isues.200 Alteration of the target sites of antimicrobials is a common mechanism of such genetic mutation.201 There are a number of reviews which provide insight into how some clinically resistant bacteria acquire antimicrobial resistance by modifying specific genes which affect the viability of the antibiotic’s mechanism of action.201–205
As S. aureus strains that were resistant to 135C were generated, a comparison of the core genome of these mutants and their corresponding wildtype strains may show any changes in specific genes. If these genetic alterations are found, the associated changes may potentially offer information on the target site of 135C.
Genomic DNA extraction, library preparation and whole genome sequencing (WGS) of eight S. aureus strains (4 generated mutants, 4 wildtype) was performed as previously described.206 Genomes were sequenced to a depth of 48X using Illumina sequencing by synthesis chemistry. To ascertain the genetic differences between mutant and wildtype strains, core genome single nucleotide variant (SNV) analysis was performed using a bioinformatics pipeline originally developed by the Sanger Institute for transmission analysis of S. aureus,207 and further refined by Daniel R Knight during his PhD studies of Clostridium difficile.206,208
WGS data was mapped to the highly annotated chromosome of S. aureus reference strain MRSA-252‡‡‡‡‡ (Genbank accession BX571856), to a depth of 43X. High-quality bona fide SNVs were detected in all four S. aureus mutant strains, relative to their wildtype counterparts (Table 3-8).
‡‡‡‡‡ Suitable reference previously used in genome mapping.447 62 | Chapter 3: Biological Evaluations of 135C
3.4 Antimicrobial resistance studies | 63
3.4.5 .1 Cell wall teichoic acids Cell wall teichoic acids (WTAs) are found embedded within the cell wall of most Gram- positive bacteria (Figure 3-3).209 They are anionic glycol (or ribo-) polymers (Figure 3-4) which can constitute up to 60 % w/w of the cell wall;210 they also play a vital role in shaping cells, cell division regulation and other physiological characteristics of Gram- positive bacteria.210,211
Figure 3-3. The Gram-positive cell wall.209
Figure 3-4. Chemical structure of WTA polymers in B. subtilis and S. aureus.210
Teichoic acid biosynthesis in Bacillus subtilis has been well characterised since the early 90s,212–214 and was shown to be an essential component of the cell wall. However, it was not until 2004 that WTAs synthesis as an antimicrobial drug target was first proposed.215 In the following years, further developments on the genomic characterisation,216 as well as the function, biosynthesis and inhibition of WTAs in B. subtilis and S. aureus were made.210,217 64 | Chapter 3: Biological Evaluations of 135C
3.4.5.2 Alterations in tag genes of 135C-resistant S. aureus strains Of the four 135C-resistant strains, three contained mutations within the teichoic acid glycerol (tag) genes, tagH, tagA, and tagG. These three genes are involved in the protein products associated with teichoic acid biosynthesis (tagA), and their ATP- binding cassette (ABC) transporter subunits (tagH and tagG). These changes suggest that WTAs may be the target site of 135C or that the antimicrobial agent plays a role in the disruption of the regular function of WTAs.
As outlined in Table 3-9, the changes in all three tag genes were nonsynonymous substitutions. These missense mutations (single nucleotide alterations) result in codon changes that lead to different amino acids.218 From a structural perspective, the changes in the amino acid side chains (highlighted in red) may be significant if the tag genes are indeed the binding sites of 135C.
The amino acid changes in the methicillin-susceptible strains, S. aureus 3784546E and NCTC 10442, both involved an arginine (86) mutation. In both cases, the mutation led to amino acids with shorter (less flexible), more hydrophobic and non-polar side chains (glycine 87 and isoleucine 88). The methicillin-resistant S. aureus RPH 15913 contained a mutation in which the amino acid changed from an asparagine (89) to a lysine (90). The side chain characteristic changes are in contrast to what was observed in the MSSA strains; the lysine side chain contains a longer (more flexible) and less hydrophobic moiety than asparagine. But similarly, the mutation provided a less polar amino acid.
The polarity of the amino acid changes may be a significant factor if WTAs are the binding sites of 135C. Structurally, 135C is a symmetrical molecule with three polar carboxylic acid moieties. It is therefore plausible that a change in the polarity of an amino acid in WTAs will have an effect on the ability of 135C to hydrogen bond to a potential binding site.
3.4.5.3 3.4.5.3 Other genomic alterations in 135C-resistant S. aureus strains When a mutation occurs without a change in its amino acid sequence, it is not likely to have a downstream effect on the end product of the gene. However, the altered sequence 3.4 Antimicrobial resistance studies | 65
66 | Chapter 3: Biological Evaluations of 135C may be recognised differently by other trans-activators (proteins acting as transcription factors) in the system which may have an effect on other cellular processes.
The mutated S. aureus strains, 3784546E and ATCC 29213, both contain base-pair alterations that are not caused by missense mutations. In S. aureus 3784546E, a base- pair mutation was detected at the locus tag SAR0512, which correspond to the ftsH gene. The ftsH gene relates to a cell division protease product in MRSA-252. In S. aureus ATCC 29213, three base-pair alterations were detected in positions that are not well characterised in the S. aureus species but are classified as a putative glycosyl transferase, a conserved methyltransferase and a putative transport protein.
It is interesting to note that S. aureus ATCC 29213 (oxacillin-sensitive MRSA) is the only strain without a change in a tag gene. A plausible explanation may be that its genetic profile associated with WTAs has already been affected by methicillin and oxacillin in the wildtype strain. The mechanism of action of these β-lactam antimicrobials involves the inhibition of penicillin-binding proteins (PBPs) which are essential in the synthesis of the peptidoglycan layer of the cell wall.219 Since WTAs are embedded in the peptidoglycan layer, the resistance mechanism of MRSA may have also targeted these sites. However, 135C was effective against MRSA, it is therefore likely that WTAs are not the only target of 135C. It is also important to note that WTAs are classified as virulence factors,210,220 and as such, 135C may have a downstream effect on the ability of Gram-positive bacteria to cause disease.
3.5 Final remarks on the biological evaluation of 135C We conducted a small pharmacological study on 135C, determining a good amount of data on its activity and toxicity profile. Unfortunately, resistance studies with the compound have shown that S. aureus can rapidly develop stable resistance against 135C, after just one passage. Subsequently, the genomic DNA of the generated resistant S. aureus strains was sequenced and genetic mutations in these mutant strains were identified. Three out of the four generated mutants presented changes in tag genes which are associated with wall proteins for teichoic acid transport and biosynthesis. This is a significant finding as it alludes to the possibility that 135C interacts with WTAs as part of its mode of action. 3.5 Final remarks on the biological evaluation o f 1 3 5 C | 67
68 | Chapter 4: Investigations into the Modes of Action of 135C
Chapter 4: Investigations into the Modes of Action of 135C 4.1 Mode of action overview Broadly speaking, there are four categories of modes of action for current antimicrobials may target: (1) cell wall synthesis, (2) protein synthesis, (3) membrane synthesis/function, and (4) nucleic acid synthesis. There are several different experiments that can be conducted to determine how a compound may or may not affect a bacterium. In the following sections are experiments that were conducted as part of this investigation to shed light on the modes of action of 135C.
4.2 Leakage and lysis experiments The inhibition of cell wall synthesis and the damage of cell membrane integrity are two of the major modes of action for current antimicrobials.221 Experiments to quantify cell lysis and the leakage of intracellular contents are often performed to determine whether novel antimicrobials have these mechanisms.222
4.2.1 Cell lysis One of the ways that an antimicrobial can kill bacteria is by affecting cell walls, and cell lysis can be monitored by measuring the optical density of an antimicrobial-treated suspension at 600 nm.223 A decrease in optical density from time = 0 to time = 240 min suggests that cells have broken apart and the suspension becomes less optically dense. Experiments were conducted at 2 (4×MIC), 32 (MBC) and 320 μg/mL (10×MBC) concentrations of 135C. There was no decrease in OD600, indicating no cell lysis (Figure 4-1).
Figure 4-1. Mean optical density of S. aureus NCTC 6571 treated with 135C in MHB at concentrations of 2 (4×MIC), 32 (MBC) and 320 (10×MBC) μg/mL.
4 . 3 T i m e - kill studies | 69
4.2.2 Cell leakage Another way that an antimicrobial can kill or inhibit bacteria is by damaging the bacterial cell membrane. Similar to cell leakage, this phenomenon can be monitored by measuring the optical density of the cell-free filtrate at 260 nm.223 An increase in optical density from time = 0 to time = 240 min suggests that nucleic acids, which absorb strongly at 260 nm, from inside the bacterial cell have leaked out.
Similar to cell lysis, experiments investigating whether exposure to 135C resulted in the leakage of intracellular contents did not show appreciable change in OD260, indicating that the cell membrane likely remained intact. These experiments suggest that bacterial cell wall and membrane damage (data not shown) are unlikely to result from the action of 135C.
4.3 Time-kill studies Time-kill experiments were performed with S. aureus NCTC 6571 to investigate whether the activity of 135C was bacteriostatic rather than bactericidal, since the MIC and MBC significantly differed from each other for this strain and other isolates of the species. The viability of S. aureus NCTC 6571 exposed to 135C at several concentrations was quantified. At 2.0 μg/mL (4×MIC), 135C did not inhibit growth. At 32 μg/mL (MBC), growth was again similar to the control from t = 0 to t = 30, however, growth was moderately inhibited after 30 minutes. At 320 μg/mL (10×MBC), viability remained similar to time zero, indicating that growth was inhibited but that cell death did not occur (Figure 4-2). At 24 h, the cells treated with 135C at 320 μg/mL had increased to a cell density of 105 CFU/mL.
Figure 4-2.Time-kill experiments of S. aureus NCTC 6571 with 135C at concentrations of 32 and 320 μg/mL. 70 | Chapter 4: Investi gations into the Modes of Action of 135C
4.4 Synergy experiments Once the MIC and MBC values of an antimicrobial were determined, experiments to detect synergistic interaction with antibiotics with known modes of action were conducted. Synergy is defined as “requiring a four-fold reduction in the MIC of both antibiotics in combination, compared with each used alone”.224 For example, if an antimicrobial is known to inhibit bacteria by affecting a particular cell pathway and its conjunctive use with 135C decreases the MIC by four-fold or more, then they are considered synergistic. There are many examples of antimicrobial combinations that have been shown to be synergistic, and synergistic interactions have the potential to shed light on the mode of action of the antimicrobial. Examples include compounds that are active against the cell wall enhancing the uptake of aminoglycosides, as well as combinations of agents active on the cell wall.225 By observing the change in antimicrobial activity across a panel of different antimicrobial agents in conjunction with 135C, it was hoped that a better understanding of the mode of action of 135C would be gained. 224
Synergy between different antimicrobial compounds can be determined using the checkerboard method where multiple concentrations of two different compounds are tested. Antimicrobial synergy data was generated using oxacillin, gentamicin, erythromycin, vancomycin, rifampicin and ciprofloxacin. Figure 4-3 shows a series of isobolograms which illustrate the result of the assays. The X-axis of the isobologram represents the dose of varying antimicrobial, and the ordinate represents the dose of 135C. 4.4 Synergy experiments | 71
Figure 4-3. Isobolograms of MICs of 135C and antimicrobial. The dotted gradient represents the theoretical line of additivity.
The fractional inhibitory concentration (FIC) indices were calculated to determine whether the combinations of antimicrobial/135C were considered synergistic, additive, indifferent or antagonistic (Table 4-1). The interaction was deemed synergistic if the FIC was ≤ 0.5, additive if the FIC was between 0.5 and 1.0, indifferent if the FIC was between 1.0 and 2.0 and antagonistic if the FIC was >2.0.226,227 72 | Chapter 4: Investigations into the Modes of Action of 135C
Table 4-1. Minimum and maximum FIC indices of antimicrobial/135C combinations.
Antimicrobial FICmin FICmax Interaction Ciprofloxacin 0.5 1.125 Additive Erythromycin 0.376 1.125 Additive Gentamicin 0.375 1.125 Additive Oxacillin 0.531 1.015 Additive Rifampicin 0.375 1.125 Additive Vancomycin 1 2.25 Indifferent * Structures of selected antimicrobials in Figure 1-6.
Although there were several FICmin values under 0.5, the results showed largely additive and indifferent effects for all six antimicrobials, indicating negligible synergistic activity with 135C. The data do not indicate any observable synergy or antagonism and as such, did not provide any further information to assist in determining the mode of action of 135C.
4.5 Azide-tagged compound for confocal microscopy Cyclooctyne-based molecules have commonly been used as probes in sensitive real- time imaging applications; highly conjugated compounds become fluorescent when their very activated and strained alkyne moiety undergoes a Click reaction (Husigen Reaction) with azides to form triazole functional groups.228 If compound 135C could be synthesised with an azide component within its structure without compromising its antimicrobial activity, this fluorescent tag could be used to determine where the compound is localised within a bacterial cell, if the cyclooctyne probe could bypass the cell membrane/wall to react with the antimicrobial agent.
Originally, we envisioned a compound where an azide tag could replace one of the carboxylic acid groups on compound 135C, such that the molecule would still retain its rigid conformation (91) (Scheme 4-1). If the synthesised compound was shown to have antimicrobial activity comparable to 135C, the treated cells could be fixed and then subjected to a solution of cyclooctyne (92). The impending Click reaction between the azide and the alkyne functional groups would allow visualisation of the fluorescent maker 93 by confocal microscopy. By following its cellular uptake, we should be able to determine where the compound locates within the cell after treatment. 4 . 5 A z i d e - tagged compound for confocal microscopy | 73
Scheme 4-1. Click reaction for fluorescent marker (93).
To synthesise compound 91, several extra steps would have to be taken before and after the key bond-forming Heck cross-coupling reactions.
4.5.1 Synthesis of azide analogue of 135C 4.5.1.1 Original envisioned synthetic pathway
Scheme 4-2. Retrosynthetic analysis of generic structure 94.
A retrosynthetic analysis of generic structure 94 (Scheme 4-2) suggests that the molecule can be constructed via two consecutive Mizoroki–Heck cross-coupling reactions from an aromatic core that contains two halide substituents with different reactivities (95). It was predetermined that 1,3,5-tribromobenzene (53) would be a good starting point for the preparation of 95. Upon treatment with 2.2 equivalence of iodine, in the presence of t-BuLi ,3,5-diiodobromobenzene (96) was accessed from 53, as described by Aida et al229 (Scheme 4-3). The consumption of the starting material was 74 | Chapter 4: Investigations into the Modes of Action of 135C confirmed by the disappearance of its single 1H NMR signal at δ = 7.61 ppm and presence of two aromatic signals at δ = 7.99 ppm (doublet) and δ = 7.82 ppm (singlet). On closer inspection of the spectrum, another set of signals can be seen at the same chemical shift, suggesting that 3,5-dibromoiodobenzene (97) was also present. TLC analysis showed that the two compounds have the same retention factor and would most likely be not separable by flash silica chromatography, and preparative TLC would not be a viable nor efficient method as a large amount of 96 was required for the synthesis.
To tackle this problem, four reactions were conducted with changes made to the reaction time in the first step of the synthesis where the halogen-lithium exchange takes place; the time was increased from 45 minutes to 1, 1.25, 1.5 and 2 hours. However, after the subsequent iodation, NMR spectra showed that the mixed iodinated compounds 96 and 97 were still present. At this point, an alternate pathway was pursued.
Scheme 4-3. Iodation of 1,3,5-tribromobenzene (53).
4.5.1.2 Alternate pathway to Azide analogue 91 The devised alternate pathway to access the azide analogue (91) begins with commercially available 3,5-dibromophenol (98) for the eventual synthesis of an ester- coupled triflic benzene (See scheme 4-5, 99). In this case the triflate can act as a pseudohalide in further palladium-mediated coupling reactions. Prior to the treatment of 98 with the ester styrene (54), a silyl protection was carried out to ensure that the phenol group would not hinder and subsequent Heck cross-coupling reaction. Baker and Phillips’ general procedure230 for the protection of phenols using DMAP, imidazole and t-butyldimethylsilyl chloride successfully installed a silyl protecting group on 98 in 83 % yield (Scheme 4-4, 100). The subsequent Heck cross-coupling reaction with 54 afforded the phenol 101 in 73 % yield. Considering the 1H NMR spectra of the compound, it becomes immediately apparent that a hydroxyl group was present by a broad signal at δ = 5.51 ppm. The three signals corresponding to the terminal alkene of the styrene substrate have shifted downfield to two doublets between δ = 6.99-7.09 ppm, 4 . 5 A z i d e - tagged compound for confocal microscopy | 75 which confirms the success of the cross-coupling reaction. A small amount of the silyl- protected compound 102 was also isolated from the reaction mixture which corresponded to less than 11 % yield. The presence of two alkane signals at δ = 1.03 and δ = 0.25 ppm, along with the downfield shift of the alkene protons as above, assisted in the confirmation of the compound.
The deprotection of silyl ethers is usually performed in the presence of Brønsted acid, Lewis acids or fluorinating agents.231 It was expected that a specific deprotection step had to be performed to remove the TBDMS group after the completion of the Heck cross-coupling reaction. However, the catalytic conditions seemed to have removed the silyl group without a great decrease in yield compared to earlier work.232,233 As such, subsequent synthesis of 101 was carried out straight from 98 in a high yield of 89 %.
Reagents and conditions: i) DMAP (10 mol%), imidazole (1.6 equiv), TBDMSCl (1.1 equiv.), DCM, 18 h, RT; ii) methyl 2-(4-vinylphenyl)acetate (2.2 equiv.), Pd2(dba)3CHCl3 (10 mol%), tBu3PBF4H (10 mol%), Cy2NMe (3 equiv.), DMF; iii) methyl 2-(4-vinylphenyl)acetate (2.2 equiv.), Pd2(dba)3CHCl3 (5 mol%), tBu3PBF4H (5 mol%), Cy2NMe (2.5), DMF.
Scheme 4-4. Silyl-protection of 3,5-dibromophenol (98) and subsequent Heck cross- coupling reaction.
The coupled phenol (101) was next treated with triflic anhydride in the presence of pyridine for the installation of a triflate group at the 1 position, which can then be used as point of attachment for a third and alternative cross-coupling reaction (Scheme 4-5). The procedure234 provided triflate 99 in a yield of 70 % and its structure was confirmed by the presence of a signal at δ = -73.19 ppm in the 19F NMR spectra, and the absence of the hydroxyl proton in the 1H NMR spectra.
76 | Chapter 4: Investigations into the Modes of Action of 135C
Scheme 4-5. Synthesis of coupled triflate (99) from coupled phenol (101).
Prior to the final cross-coupling reaction, the azide styrene substrate (103) was synthesised by treating 4-vinylbenzylchloride (55) with sodium azide under the conditions described by Yoon and Williamson (Scheme 4-6).235 The procedure afforded the desired compound 103 in 88 % yield and its identity was confirmed by the downfield shift of the methylene carbon resonance from δ = 46 in compound 55 to 55 ppm in the product 13C NMR spectra.
Hopeful that this last cross-coupling reaction will be the penultimate step for the azide- tagged analogue 91, the reaction was performed using the coupled triflate 99 and the azide styrene 103 under the previously established Heck cross-coupling conditions for compound 104. Unfortunately, the desired product was not synthesised and starting material 99 was recovered (Table 4-2, entry 1). Other Pd2(dba)3∙CHCl3 conditions with different ligands and bases were trialled with no success (Table 4-2, entry 2-4); changing the catalyst:ligand ratio (Table 4-2, entry 5) and increasing the loading of the catalyst, ligand and base (Table 4-2, entry 6) also did not induce a reaction.
t Reagents and conditions: Pd2(dba)3CHCl3 (5 mol%), Bu3PBF4H (5 mol%), Cy2NMe (2 equiv.), DMF, 80 °C. Scheme 4-6. Synthesis of azide styrene 103 and subsequent cross-coupling with triflate 99. 4 . 5 A z i d e - tagged compound for confocal microscopy | 77
4.5.1.3 Reactivity of triflate 99 and azide styrene 103 Several other palladium catalysts were trialled for their reactivity with 99 for the synthesis to 104. Several conditions using palladium acetate were trialled with different ligands (or in absence of, Jeffery’s condtions) and bases (Table 4-2, entries 7-10),236,237 with no desired product synthesised; the Hermann–Beller catalyst 105 (Table 4-2, entry 10)238 and palladium tetrakis triphenylphosphine (Table 4-2, entry 11)239 were also unsuccessful in the C-C cross-coupling reaction. For all of the conditions trialled, the triflate 99 was recovered at the end of the experiment. It is assumed the pseudohalide - OTf was not a good enough leaving group combined with the richly electron dense conjugated aromatic system.
At this point, the reactivity of the azide styrene 103 towards simple 4-methoxy phenyl triflate 106 was tested and was found unfruitful for the synthesis of 107 (Scheme 4-7). A literature search revealed that this azide styrene 103 has never been reported as a Mizoroki–Heck cross-coupling partner. The majority of the reactions reported are [2+3]-cycloadditions at the azide moiety. Styrene 103 was also not able to be recovered after the test reaction.
Reagents and conditions: Pd2(dba)3CHCl3 (5 mol%), tBu3PBF4H (5 mol%), Cy2NMe (2), DMF, 80 °C. Scheme 4-7. Attempted cross-coupling reaction of azide styrene 103 and phenyl triflate 106.
To further determine the reactivity of triflate 99, the compound was used to couple with the styrene ester substrate 54, as well as the 4-vinylbenzylchloride (55). However, neither reaction was successful. In a last attempt, triflate 99 was subjected to Sonagoshira cross-coupling conditions with phenylacetylene (108) without success. The cross-coupling of triflate 99 was not pursued any further at this point, and another plan was devised for the synthesis of an azide tagged analogue.
78 | Chapter 4: Investigations into the Modes of Action of 135C
4 . 5 A z i d e - tagged compound for confocal microscopy | 79
Table 4-3. Reaction profile of triflate 99 with different coupling partners.
Entry Styrene Pd catalyst Ligand Base Solvent % (mol%) (mol%) (equiv.) yield
1 Pd2(dba)3CHCl3 tBu3PBF4H Cy2NMe DMF n.r. (10) (20) (2.5)
2 Pd2(dba)3CHCl3 tBu3PBF4H Cy2NMe DMF n.r. (10) (20) (2.5)
3 Pd2(PPh3)2 (10) CuI (10) Et3N (2) THF n.r.
4.5.1.4 Alternative azide analogue Since the coupled phenol 101 could be readily accessed synthetically (Scheme 4-4), an ether analogue structure of the azide-tagged compound was envisioned (Scheme 4-10, 109). Using an appropriate halogen coupling partner, phenol 101 could be transformed via a Williamson ether synthesis.
Ether 110 was synthesised by treating phenol 101 with potassium carbonate in the presence of α,α′-dichloro-p-xylene (111) in DMF; this reaction gave this novel compound 110 in 31% yield and two extra sets of aromatic doublets (δ = 7.49 and 7.43 ppm) in the 1H NMR spectrum confirmed the reaction’s success. The two singlets at δ = 5.11 and δ = 4.60 ppm were assigned to the two alkyl protons adjacent to the ether oxygen, and the two adjacent to the chloride, respectively. The doublets at δ = 7.43 ppm (J = 8.3 Hz) and δ = 7.49 (J = 8.2 Hz) were assigned to the aromatic protons from the 80 | Chapter 4: Investigations into the Modes of Action of 135C newly attached α,α′-dichloro-p-xylene, with the further downfield set corresponding to the protons closest to the ether linkage. It is noted that 31 % is a very low yield for a Williamson ether synthesis as alkoxide ions formed from phenols are highly reactive. A likely explanation for this low yield of the desired product 110 could due to a subsequent reaction with another unit of the phenol (Scheme 4-8), forming a diether 112.
Scheme 4-8. Formation of diether 112.
To tackle this low yield, an alternative pathway was designed where one chloride unit on 111 was displaced by an azide group before the ether synthesis (Scheme 4-9). Unfotunately, the azide displacement on 111 occurred on both chloride atoms. As the mono- and di-azido compounds had almost identical retention factors, the desired mono-azido compound 113 was not isolated.
Conditions: (i) NaN3 (1.0 equiv.), DMF, 60 °C, 16 h; (ii) K2CO3 (2.0 equiv.), KI (cat.), acetone, 75 °C, 16 h. Scheme 4-9. Alternate synthetic route to azide-tagged compound 109.
Reverting back to the previous synthesis, the remaining chloride atom in ether 110 was displaced by an azide group upon stirring in the presence of sodium azide in DMF. The 4 . 5 A z i d e - tagged compound for confocal microscopy | 81 desired product 114 was obtained in 65 % yield, first revealed by a characteristic strong azide stretching absorbance at 2095 cm-1 in the IR spectrum. In the 1H NMR and 13C
NMR spectra, the signals assigned to the CH2 group adjacent to the chloride atom in 110 shifted from δ = 4.60 ppm to δ = 4.37 ppm, and from δ = 45.94 to δ = 54.62, respectively.
Reagents and conditions: (i) 1,4-bis(chloromethyl)benzene (1.0 equiv), K2CO3 (2.0 equiv.), KI (cat.), acetone, 75 °C, 16 h; (ii) NaN3 (1.0 equiv.), DMF, 60 °C, 16 h; (iii) LiOH.H2O (4 equiv.), EtOH/H2O (1:9), 16 h. Scheme 4-10. Synthetic route to azide-tagged compound 109.
Lastly, a saponification was performed on 114 to remove the methyl ester groups using an excess amount of lithium hydroxide in ethanol/water under reflux; the reaction produced 109 in 89 % yield. The successful removal of the methyl ester groups was confirmed by the absence of the methyl proton signal originally at δ = 3.72 ppm in the 1H NMR spectrum, as well as the methyl carbon signal originally at δ = 52.22 ppm in the 13C NMR spectrum. It is important to note that the isolated pale yellow solid had 1 low solubility; therefore, the H NMR spectrum was collected at 50 °C in methanol-d4. A high resolution molecular ion was also found at [M+H] = 560.2185, matching the predicted value.
82 | Chapter 4: Investi gations into the Modes of Action of 135C
4.5.1.5 Antimicrobial activity of azide analogue 109 To determine whether the synthesised analogue 109 was suitable for investigating cell localisation and mode of action, it first had to be active against the bacteria of interest. The MIC value of 109 was determined to be 1 µg/mL against S. aureus NCTC 6571, which is three-fold higher than the MIC value of 135C (0.25 µg/mL). This decrease in activity was expected as the azide compound has one less carboxylic acid substituent, the assumed pharmacophore of 135C. The MBC of 109 was greater than 64 µg/mL.
4.5.2 Fluorescent microscopy of azide-tagged 135C with S. aureus NCTC 6571 (imaging and data processing performed by Associate Professor Paul Rigby) A 4-dibenzocyclooctynol (DIBO) was chosen as the fluorescent probe as it was previously used within the Stewart group and was shown to be successful in reacting with azide-tagged thalidomide derivatives.240 Additionally, Olia et al. have shown that when this F1-DIBO (115) undergoes a Click reaction with an azide-functionalised ethoxylamine glucosylnitroxide, the resulting compound (116) can permeate P. aeruginosa biofilms241 (Figure 4-4). P. aeruginosa is a Gram-negative bacterium which has an extra lipopolysaccharide outer membrane (Figure 4-4).242 It is then a conjecture that F1-DIBO is likely to be able to permeate Gram-positive bacteria such as S. aureus, as it is smaller in structure and its target does not have an extra outer membrane that the compound has to pass through. Therefore, it was hoped that F1-DIBO could be used as a stain after the cellular uptake of 135C in S. aureus. This fluorescent probe F1-DIBO was prepared by Dr. Charles Heath, following the synthesis described by Friscourt, Fahrni, and Boons.243
Figure 4-4. Structure of the 4-dibenzocyclooctynol (F1-DIBO)243 115 and ethoxylamine glucosylnitroxide triazole 116.241
4 . 5 A z i d e - tagged compound for confocal microscopy | 83
Three negative controls and one sample were prepared on microscope slides by heat- fixing 135C(N3) (109) - treated or untreated cells onto glass coverslips, stained with a F1-DIBO methanol solution (samples C & D, only), before applying mounting media and closed onto a microscope slide. The four prepared slides are as follows:
A. S. aureus; (F1-DIBO and 135C(N3) negative control)
B. 135C(N3)-treated S. aureus; (F1-DIBO negative control)
C. S. aureus stained with F1-DIBO; (135C(N3) negative control)
D. 135C(N3)-treated S. aureus stained with F1-DIBO Images were taken with a Nikon Ti-E inverted motorised microscope with a Nikon A1Si spectral detector confocal system running the NIS-C Elements software. Confocal, transmission and their overlayed images of each slide are as shown in Figure 4-5. 84 | Chapter 4: Investigations into the Modes of Action o f 135C
Confocal image Transmission image Overlayed image
A
B
C
D
Figure 4-5. Confocal, transmission and overlayed images of samples. A) S. aureus; B) 135C(N3)-treated S. aureus; C) S. aureus stained with F1-DIBO; D) 135C(N3)-treated S. aureus stained with F1-DIBO 4 . 5 A z i d e - tagged compound for confocal microscopy | 85
Unstained samples A and B shows auto-fluorescence of the bacterial cells, and it appears that the F1-DIBO stain muted the autofluorescence in sample C. Sample D shows a similar fluorescent level as A and B, however, if the F1-DIBO stain can mute autofluorescence, then it is speculated that the fluorescence detected in the sample may be due to the interaction between 135C-N3 and F1-DIBO, and not autofluorescence. However, further imaging studies will have to be performed to confirm this speculation.
If the fluorescence is indeed caused by bacterial autofluorescence and not the interaction between 135C-N3 and F1-DIBO, then it would be fair to conject that either F1-DIBO was not able to pass through the cell wall and membrane, or that 135C-N3 had been degraded during the cellular uptake. The prior theory may be more likely because even if the structure of 135C-N3 (109) is degraded, free azide should still be in the cell for the impending Click reaction, unless it was washed out during the sample preparation process. The intense fluorescence detected in sample D that are outside of the bacterial cells are presumed to be precipitants of the Clicked fluorophore (Figure 4-6, 117).
Figure 4-6. Structure of Clicked fluorophore (117).
It is interesting to note that the 135C-N3-treated S. aureus colonies (B and D) are aggregated to each other in a clumping formation. This phenomenon has been reported previously to be caused by bactericidal catechins and is a response to hostile environments when damage to the lipid bilayer is caused.244 By decreasing cell surface area, bacterial cells will absorb less substrates in its immediate surroundings,245 hence 86 | Chapter 4: Investigations into the Modes of Action of 135C effectively reducing the concentration of the uptake of antimicrobial substances. This activity implicates that cytoplasmic membrane is a possible target site for 135C-N3’s activity. Although this membrane-related aggregation of bacterial cell is supported by other studies in the literature,246,247 there are other environmental stresses that may cause the aggregation of bacterial cells.248–250 Therefore, further studies into this phenomenon are required to elucidate the cause of the aggregation. 4 . 5 A z i d e - tagged compound for confocal microscopy | 87
88 | Chapter 5: Summary and Conclusions - P a r t I
Chapter 5: Summary and Conclusions- Part I The first part of this thesis described the synthesis and biological evaluation of antimicrobial compound 135C. The overarching aim of the project was to determine the mode of action of 135C as well as to establish its biological profile. One of the goals was to synthesise analogues via carboxylic acid group replacement to determine whether changing these moieties will address the problem of decreased antimicrobial activity in the presence of protein. Six analogues were successfully synthesised and isolated (Figure 5-1); however, the low solubility of some of these candidates imposed limitations on their viability in biological assays. The interaction between protein serum albumin and 135C was confirmed by way of saturation transfer NMR experiments, which indicated that BSA binds 135C towards the methylene protons, signifying that the binding site is at the carboxylic acid moiety.
Figure 5-1. Core structure of 135C and corresponding analogues
A variety of parameters were determined for the biological evaluation of 135C. From the in vitro susceptibility studies, the spectrum of activity of 135C was determined against a variety of aerobic Gram-positive bacteria in the Staphlococci, Streptococci, and Micrococci genera. The susceptibility tests showed that 135C has good antimicrobial activity towards these species with MIC values as low as 0.12 μg/mL. However, MBC values were >32 μg/mL and this large difference between the MIC and MBC values suggests that the activity of 135C is bacteriostatic and not bactericidal.
Compound 135C was found to be less active towards anaerobic Gram-positive bacteria, and under anaerobic conditions, it was found to be more active against S. aureus NCTC 6571 with a 14-fold decrease in MIC, suggesting that the compound may be oxidised to 4 . 5 A z i d e - tagged compound for confocal micr o s c o p y | 89 a less active species when incubated in the presence of oxygen. Apart from M. catarrhalis, 135C did not exhibit antimicrobial activity towards Gram-negative bacteria, except when assays were performed with PMBN, indicating that the outer membrane is involved in susceptibility.
Compound 135C was found to present no mutagenic and low haemolytic activity, which is a positive characteristic for potential drugs for human consumption. However, S. aureus strains were able to develop a stable resistance towards the compound in a very short time period, rendering its potential as a viable therapeutic inadequate for the current pharmaceutical market where antibiotic resistance is already a major problem.
A comparison of the core genome sequence of the generated mutants and their wildtype counterparts revealed a change in teichoic acid-associated tag genes. Changes in these genes allude to an interaction of 135C with wall teichoic acids as part of its bacteriostatic action, perhaps even affecting the pathogenicity of Gram-positive bacteria since WTAs are also considered to be virulence factors.
Time-kill studies indicated that 135C has a relatively slow effect on S. aureus and studies investigating cell lysis found no evidence for this effect. Experiments investigating whether 135C caused cell leakage gave a negative result as bacterial cell contents were not detected in cell-free filtrates. These experiments have shown that bacterial cell wall damage is unlikely to be a mode of action of 135C. It was also found to show negligible interaction, be it synergistic or antagonistic, with six different antibiotics.
As a chemical approach, an azide-tagged analogue of 135C was synthesised to be used as a fluorescent tag precursor to determine the location of the compound within a bacterial cell after its cellular uptake. 135C-N3 (109) was synthesised over four steps from 3,5-dibromophenol (98) and ester styrene (54) with an overall yield of 16 % (Scheme 5-1). 90 | Chapter 5: Summary and Conclusions - P a r t I
Scheme 5-1. Synthesis of azide-tagged 135C analogue, 135C-N3 (109).
135C-N3 (109) was found to be active against S. aureus and was stained with the 4- dibenzocyclooctynol, F1-DIBO (115) after cellular uptake for the fluorescent imaging probe (117). Unfortunately, the experimental sample showed only autofluorescence in the confocal image. However, it was found that S. aureus colonies aggregate together when treated with 135C-N3 (109), suggesting that cytoplasmic membrane may be a possible target site for this compound’s activity.
In conclusion, a good biological profile of 135C was established and its interaction with serum albumin was confirmed. The mode of action of 135C is still unclear, however, genetic mutations of S. aureus caused by the compound have shed light on the possibility that 135C interacts with cell wall teichoic acids. This direction towards the cell wall and membrane could be explored further to identify the mechanism of this interaction. 4 . 5 A z i d e - tagged compound for confocal microscopy | 91
92 | Chapter 5: Summary and Conclusions - P a r t I
4 . 5 A z i d e - tagged compound for confocal microscopy | 93
PART II: NEW NICKEL CATALYSTS AND THEIR APPLICATIONS IN ORGANIC SYNTHESIS 94 | Chapter 6: General Introduction - P a r t I I
Chapter 6: General Introduction- Part II 6.1 The Importance of the cross-coupling method in organic synthesis At the heart of chemistry is synthesis. Historically, stoichiometric routes were used in the synthesis of fine chemicals, which not only has low atom efficiency and high monetary costs, it also utilises more materials which can lead to a higher environmental impact. The discovery of Grignard reagents at the turn of the 20th century revolutionised the approaches of synthetic chemistry by the introduction of organometallic compounds playing a major role in both small-scale syntheses and industry.251 The use of transition metal-based processes contributed to important economic and environmental considerations such as alleviating the pressure on production costs and minimising waste in industry.252 Catalytic routes in the industrial production of chemicals are, in general, more economical and less environmentally stressing; they are now widely preferred over stoichiometric routes whenever possible. That said, there are other considerations such as the cost of multistep syntheses, in which the exponential cost of catalysis may be higher than using traditional stoichiometric routes (Figure 6-1).253
Figure 6-1. Use of transition metals in chemical synthesis.252
The role of transition metals in organic synthesis is wide and varied, and a plethora of chemical reactions can now be carried out in their presence. Some prominent early examples include hydrogenation, isomerism, hydrosilylation and oxidation, to name a few.254–260 However, the formation of new carbon-carbon and carbon-heteroatom bonds in organic synthesis is one of the most important processes in the construction of complex molecules from simple precursors. Accordingly, the use of transition metals in these methods and applications have been widely researched for direct bond formations between sp and sp2 C-atom centres; especially the carbon-carbon bond formation between unsaturated species.261,262 In the past century, the development of 6.2 Notable breakthroughs in metal - catalysed cross - coupling reactions | 95 methodologies in this field has allowed the emergence of cross-coupling reactions as one of the most powerful synthetic techniques in organic chemistry.
6.2 Notable breakthroughs in metal-catalysed cross-coupling reactions In this section, a selection of the most significant breakthroughs in the field of cross- coupling chemistry for carbon-carbon bond formation is presented.
6.2.1 Humble beginnings As noted in the previous section, prior to the use of catalytic processes, stoichiometric preparations were used in the synthesis of many compounds. Early reports of copper, organomagnesium and organosodium-mediated processes are noted in regards to the functionalisation of alkyl and aryl halides. The first metal-mediated homocoupling was reported in 1855; in this work, Wurtz described the homodimerisation of alkyl halides (118) in the presence of stoichiometric amounts of sodium.263 Several years later, Fittig, then Tollens (with potassium), extended the work to involve the homocoupling of aryl halides (120) (Scheme 6-1).264–266 However, these early investigations had limited application due to the violently reactive nature of sodium and potassium reagents.
Scheme 6-1. Early discoveries of metal-assisted homocoupling reactions.263,264,267–269
In 1869, the first metal-mediated coupling of acetylides (122) was reported by Glaser.267 In these studies, the oxidative dimerisations of both copper and silver phenylacetylide were described, using the respective metal chlorides in stoichiometric amounts. 96 | Chapter 6: General Introduction - P a r t I I
Subsequently, the development of this metal-mediated C(sp)-C(sp) bond forming process was extended to produce C(sp2)-C(sp2) bonds by Ullmann in 1901;268 this work described the homocoupling of 2-bromo- and 2-chloronitrobenzene (125), however, the reactions required super-stoichiometric amounts of copper under harsh physical conditions.
Around the same time, the development of milder Grignard reagents was underway, but their reactivity with alkyl and aryl halides were limited and undesired side reaction detracted from the efficiency of this type of reaction.270 It wasn’t until 1914 that C(sp2)- C(sp2) bond formation was shown to be possible using Grignard reagents by Bennett and Turner, with their report on the dimerization of phenylmagnesium bromide (127) using a stoichiometric amount of chromium(III) chloride.269 A similar process with copper chloride was later reported by Krizewsky and Turner in 1919. 271
6.2.2 Discovery phase of catalytic cross-coupling reactions Even though the formation of new C-C bonds is possible using stoichiometric amounts of metal, as shown in the above examples, chemists investigated the possibility of reducing quantities of metals used to catalytic amounts. The obvious advantages of this change would improve atom efficiency, lower costs, as well as being better for the environment. As early as 1923, the first catalytic cross-coupling process was discovered by Job et al. where phenylproprionic acid (129) was synthesised from phenylmagnesium bromide (127) and ethylene (128) in the presence of nickel chloride, 272 after a CO2 treatment (Scheme 6-2). In 1939, Meerwein reported an investigation on the decarboxylative coupling of coumarins and cinnamic acids with catalytic quantities of copper(II) salts.273 Both pioneering works of Job and Meerwein were minimally acknowledged by the scientific community at the time of their publication during the inter-war period. However, these publications were the first to introduce the idea of catalysis in the field of organometallics. In 1943, Kharasch introduced cobalt as a suitable catalyst for the cross-coupling reaction between Grignard reagents (127) and vinyl bromide (133) for the synthesis of styrenes (134).274,275
6.2 Notable breakthroughs in metal - catalysed cross - coupling reactions | 97
Scheme 6-2. First reports of C-C bond formations with catalytic quantities of metal salts.272–274
Whilst the studies of Job, Meerwein and Kharasch demonstrated that catalytic quantities of transition metals could be used for carbon-carbon bond formation, their limited scope and functional group compatibility, as well as their selectivity for homocoupling over the sometimes desired cross-coupling products greatly restricts their application. This issue of selectivity for homocoupled products was addressed first in 1957 with Cadiot and Chodkiewicz’s copper-catalysed cross-coupling of bromoalkynes (135) and alkynes (136).276 This was followed by Castro and Stephens’ breakthrough in 1963 with the first selective C(sp2)-C(sp2) bond formation between aryl or vinyl halides (138) with copper acetylides (123).277 It is noted that the prior reactions required harsh conditions, but nonetheless, these were the first examples of selective C(sp)-C(sp) and C(sp2)-C(sp2) bond formation (Scheme 6-3). 98 | Chapter 6: General Introduction - P a r t I I
Scheme 6-3. The Cadiot-Chodkiewicz and the Castro-Stephens reactions.276,277
6.2.3 The rise of palladium in cross-coupling chemistry Since Wollaston’s 1802 discovery of palladium metal, little molecular use of this precious metal had been found. Apart from its use in medical instruments and as a steel substitute, its chemistry was fairly neglected due to the popularity of platinum and nickel in the redox and hydrogenation reactions of unsaturated hydrocarbons.278 However, following the discovery of the Wacker process, in which oxidative conversion of ethylene to acetaldehyde is mediated by catalytic amounts of palladium,279 palladium was established as a metal of importance for organic synthesis.
In the early 1970s, both Mizoroki and Heck independently reported the palladium- catalysed cross-coupling reaction between aryl iodide (140) with alkenyl partners (128, 134), without the use of highly toxic reagents such as mercury, tin or lead (Scheme 6- 4).280–282 These publications also showed the versatility of these palladium-catalysed reactions as the conditions were also applicable in the coupling of other aryl, benzyl, and styryl halides with olefins in high efficiency. In the years following, remarkable advancements were made in the application of the Mizoroki–Heck cross-coupling reaction in organic synthesis, from the formation of simple hydrocarbons, to the production of structurally complex natural products.283–289 Today, the Mizoroki–Heck reaction is one of the most widely-used cross-coupling methods in industry.290–292
6.2 Notable break throughs in metal - catalysed cross - coupling reactions | 99
Scheme 6-4. Palladium-catalysed Mizoroki–Heck cross-coupling reaction.280,282
Notwithstanding the rise in popularity of palladium catalysis, Corriu and Kumada concurrently presented the nickel-catalysed cross-coupling of aryl and alkenyl halides (142, 145) with Grignard reagents (143, 146) in 1972 (Scheme 6-5).293,294 In these reports, no undesired homocoupling of the substrates were observed, and E-selectivity was always favoured. These reactions were later adapted by Murahashi and Jutand to be compatible with palladium as the catalyst source.295,296
Scheme 6-5. Nickel-catalysed cross-coupling reactions of Grignard reagents.293,294
6.2.4 Improvements on palladium-catalysed processes The scope of palladium was continually extended as chemists proved its value and higher selectivity over other metals in coupling chemistry. Based on the Castro- Stephens designed C(sp2) -C(sp2) coupling of acetylenes (Scheme 6-3), Sonogashira developed a palladium-catalysed process with a copper(I) salt as co-catalyst (Scheme 6- 6).297 This greatly improved upon the original method, as it only required catalytic 100 | Chapter 6: General Introduction - P a r t I I amounts of copper and palladium, can be conducted at room temperature, and has a very high functional group tolerance which in turn dramatically expanded the scope of the reaction. The year 1976 saw Ei-ichi Negishi’s investigation of the cross-coupling of organo-aluminium and -zinc reagents (149) with aryl halides (150) which represented milder reaction conditions than previous organometallic reagents, as well as more diverse functional group compatibility.298 In the same year, Jutand showed that it was possible to use such organozinc reagents (152) for C(sp2) -C(sp3) cross-couplings.295 In 1978, Stille demonstrated the accessibility of ketones (156) from the coupling of acid chlorides (154) with organotin reagents (155).299 This was an important milestone since this other organometallic reagents have not been shown to be able to couple carbonyls in such a selective manner as the reagents are also highly reactive towards the resultant ketone. A plethora of other electrophiles are also tolerated in this reaction, however, the toxic nature of organotin reagents later rendered this catalytic process to be highly undesirable, especially in industry.300
Scheme 6-6. Palladium-catalysed cross-coupling reactions with other metals.295,297–299
6.2 Notable breakthroughs in metal - c a t a l y s e d c r o s s - coupling reactions | 101
6.2.5 Other notable key cross-coupling examples The Suzuki–Miyaura reaction published in 1979 is arguably one of the most important C-C bond formation process along with the Mizoroki–Heck reaction;§§§§§ the reaction is carried out between alkenyl or aryl halides (120) and alkenylboranes (157) under mild and convenient reaction conditions (Scheme 6-7).301,302 Subsequent studies have presented that other organoboron compounds such as arylborones, boronic acids and esters,303 alkyltrifluoroborates,304 and MIDA boronates,305 can be used in this cross- coupling process. Organoboron compounds are generally air- and moisture stable, and the by-products from this reaction are easily removed and have low toxicity. It is due to these advantageous features that the Suzuki–Miyaura reaction is a cornerstone process in organic synthesis.
Scheme 6-7. The Suzuki–Miyaura reaction.301
During the 1970s, the addition of phosphine ligands in these reactions was introduced and is now often an essential feature of cross-coupling processes. This important breakthrough was pioneered by Tamao and Kumada; the addition of these ligands allowed the cross-coupling of less reactive compounds such as aryl chlorides to be realised.306 N-Heterocyclic carbene (NHC) and amine ligands have also been shown to be used in metal-catalysed cross-coupling reactions,307–310 but it is not in the scope of this thesis to discuss them in detail.
The examples mentioned in this section are only a glimpse of the cornerstone processes in the rich history of the field of metal catalysed cross-coupling chemistry; the selected reactions only paints a general picture of the early stages in the evolution of carbon- carbon bond formation. In the past 40 years, numerous advances in the field have
§§§§§ A recent search on SciFinder (16-02-2017) revealed 19636 hits for “Suzuki reaction” and 9881 hits for “Heck reaction” 102 | Chapter 6: General Introduction - P a r t I I contributed to the ever-growing economic and environmental demands of the industry where the building blocks of life are based on such carbon-carbon bonds. Appropriately, the award of the Nobel Prize in Chemistry in 2010 to Richard Heck, Ei-ichi Negishi, and Akira Suzuki- three major contributors in the field of palladium catalysis- epitomises its role in society.311
6.3 Recent pharmaceutical applications Cross-coupling chemistry is prominent in commercial industrial processes in today’s chemical market.312 The production of many precursors and final drug entities within the pharmaceutical industry are synthesised with the aid of metal catalysts;313 this is also true for many small-scale academic pursuits in medicinal chemistry. The following are a few examples of recent pharmaceutical compounds which utilise cross-coupling reactions as key steps in their synthetic pathway.
(±)-Streptonigrin (159) is a tetracyclic aminoquinoline-5,8-dione with potent antitumour and antibiotic properties which was originally isolated from Streptomyces flocculus in 1959 by Rao and Cullen.314 The total synthesis of (±)-streptonigrin was achieved in 14 linear steps in 2013 by Donohoe et al.,315 where the key connections are a Stille cross-coupling between the quinoline and pyridine units, followed by the attachment of the lower anisole aromatic ring via a Suzuki cross-coupling (Figure 6-2).
Figure 6-2. Retrosynthetic disconnection of (±)-Streptonigrin (159)
Axitinib (160) is a vascular endothelial growth factor antagonist which is currently used as treatment for advanced kidney cancer.316 The key connections in this structure are dependent upon two palladium-catalysed cross-couplings: a Migita C-S coupling and Mizoroki–Heck reaction. Pfizer pharmaceuticals has successfully synthesised this 6.4 General mechanism of cross - coupling reactions | 103 compound at a commercial-scale of 28 kg.317 It is also interesting to note that the E- selective Mizoroki–Heck reaction in this synthetic pathway was also used as a control strategy to minimise the amount side products formed (such as homocoupled benzamide 161)****** in the entire process.
Figure 6-3. Retrosynthetic analysis of Axitinib (160)
The short and practical synthesis of the antihistamine olopatadine (162) involves the use of a stereoselective Heck cyclisation.318 Both olopatadine and its E-isomer (163) can be synthesised by this process and differentiated by the use of starting material.
Figure 6-4. Olopatadine (162) and its isomer trans-olopatadine (163)
6.4 General mechanism of cross-coupling reactions As with the development of any chemical reaction, chemists try to understand how it proceeds on a molecular level by determining its mechanism. One of the main contributors to the study of the mechanistic pathways of cross-coupling reactions was John Stille with his work on the coupling of organotin reagents.319 The research into the mechanism of a reaction is central to its success in terms of optimisation, as well as for
****** Structure of homocoupled benzamide side product: 104 | Chapter 6: General Introduction - P a r t I I the development of related novel transformations. As most of the first wave of efficient metal catalysed cross-coupling reactions discovered were catalysed by palladium, it is unsurprising that the majority of the mechanistic studies focus on these processes; nevertheless, there are also a number of studies that feature other transition metals, particularly nickel and copper.320–323 Each named cross-coupling reaction has its own unique mechanistic particularities; however, they all follow a general pattern which is described in Figure 6-5 (left).
Figure 6-5. Generalised mechanism of cross-coupling reactions (left) and a comparison with the Mizoroki–Heck reaction (right).
A highly generalised catalytic cycle is presented in the left of Figure 6-5. Broadly speaking, there are three basic steps in a catalytic cycle: oxidative addition, transmetallation and reductive elimination. Before entering the catalytic cycle, the metal species is usually reduced to its active oxidation state through the loss of ligands; for palladium and nickel catalysts, a reduction from a +II to a 0 oxidation state is most common.116 Additionally, if a palladium catalyst is in the 0 oxidation state, then simple dissociation of ligands occurs to a coordinatively unsaturated LnM(0) species. The
LnM(0) species then undergoes oxidative addition with a R-halide or R-pseudohalide 6.5 Organonickel chemistry | 105 species (R1-X), producing a M(II) complex with the first R group (R1). This complex then reacts with the second R group via a transmetallation process, and the new coupled R1-R2 bond is formed in the following reductive elimination step, which also regenerates the catalytically active M(0) species for the continuation of the catalytic cycle.
The Mizoroki–Heck reaction differs from this general catalytic cycle considerably, and involves a migratory insertion with an alkene and a syn-β-hydride elimination step which forms the new R1-alkene bond, rather than a transmetallation step and the formation of the R-R bond in the reductive elimination.324 A simplified depiction of the process is presented on the right of Figure 6-5. A more detailed discussion of the mechanistic aspects of the Heck-Mizoroki and Suzuki cross-coupling reactions is presented in chapter 7.
6.5 Organonickel chemistry 6.5.1 Historical perspectives and notable discoveries The discovery of nickel is an interesting tale. It was first found as a red mineral in a copper mine in Erzgebirge from which copper was unable to be extracted. Unbeknownst to the superstitious miners that this ore was not composed of copper, they named this red mineral kupfernickel (copper-nickel), after Nickel, a deceptive little spirit.325,326 It was not until 1751 that Swedish metallurgist Axel Fredrik Cronstedt was able to isolate a white metal from kupfernickel (or nicolite, NiAs, as it is known today) through a series of redox experiments; after observing its chemical and physical properties, he appropriately proposed this new element be named nickel.327
The Mond method is one of the first industrial processes of nickel refinement. In 1890, Mond discovered that the reaction between carbon monoxide (164) and metallic nickel 328,329 can occur at 50 °C to form toxic and volatile Ni(CO)4 (165). When this nickel tetracarbonyl reaches temperatures over 180 °C, it decomposes to form pure nickel deposits (Scheme 6-8).330 The Mond method is still in use in industry today, even though the intermediate Ni(CO)4 is exceptionally toxic to humans with an LD50 of 3 ppm over a 30-minute exposure, and an immediately fatal if exposed at 30 ppm.331 This pivitol discovery inspired many chemists to explore the chemical capabilities of nickel and led to several important advances in the field. In 1897, Sabatier and Senderens reported the hydrogenation of ethane (166) from ethylene (128) and hydrogen in the 106 | Chapter 6: General Introduction - P a r t I I presence of nickel.332 This method was then extended to show the possibility of nickel- catalysed hydrogenation of acetylene (167) to ethylene (128) in cold conditions. This seminal discovery saw Sabatier win a Nobel Prize in 1912. Around this time, the inter- war period, little attention was paid to the chemistry of organonickel.
Scheme 6-8. Early discoveries of organonickel chemistry.
The next notable breakthrough in organonickel chemistry came in 1948, when Reppe, Schlichting, Klager and Toepel prepared cyclooctatetraene (COT, 168) from the oligomerisation of acetylene (167) in the presence of nickel 169 (Scheme 6-9). The findings and speculations from this work led to the idea that the existence of a metallacycle serves as the mechanism of the reaction.333 From an industrial standpoint, Reppe made a major contribution in this field when he also discovered a series of homogeneous catalytic carbonylations using carbon monoxide, a by-product in the manufacture of acetylene.334,335
Scheme 6-9. Cyclooctatetraene formation as proposed by Reppe et al.336
The “nickel effect” was first reported in 1952 when Holzkamp observed the altered
“growth reaction” of ethylene to α-olefins catalysed by AlR3 whereby only 1-butane dimers are formed.333,337 Following this work, Ziegler and Natta developed titanium and vanadium-based catalysts (“Zeigler-Natta catalysts”) that allowed the configurational control of polymers from terminal alkenes for the synthesis of industrially important 6.5 Organonickel chemistry | 107 materials.338 This work led to new methodologies in the development of transition-metal complexes by Wilke and co-workers.
In 1959, Wilke was able to isolate a mixture of 1,5-cyclooctadiene (COD, 170), 4- vinylcyclohexene (VCH, 171), and all trans-1,5,9-cyclododecatriene (CDT, 172) from a butadiene polymerisation experiment (Figure 6-6); in this reaction, Wilke was trying to synthesise the Ziegler catalyst from nickel acetylacetonate and AlEt2OEt, rather than the commonly used NiCl2/Et2AlCl system. The subsequent mechanistic investigations greatly contributed to our understanding of several steps in catalytic cycles, as well as marking the isolation of the first nickel(0) olefin complex, Ni(CDT), and succeeding nickel olefin complexes that have been very prominent in industrial processes (Figure 6- 7).
Figure 6-6. Structures of the cyclised products obtained from butadiene polymerisation experiments.
Figure 6-7. Ni(0) olefin complexes discovered and isolated by Wilke and co-workers.
Of the nickel(0) olefin complexes that were synthesised by Wilke et al., bis(cyclooctadiene)nickel(0) (Ni(cod)2) presented as one of the most important nickel 333 catalysts (or precatlayst) in the next decades. Initially, Ni(cod)2 was used or olefin oligomerisation reactions in industry, but the exploration of its reactivity has shown it could be used to catalyse a plethora of cross-coupling reactions.339 Although the preparation of this 18-electron complex is not trivial, it is still widely used today.
Another undesirable trait of Ni(cod)2 is its high sensitivity to oxygen which demands 108 | Chapter 6: General Introduction - P a r t I I careful handling, storage, transport and precautions;340 it is for this reason that many research groups are interested in finding new air-stable nickel(0) complexes that have similar catalytic/precatalytic properties to Ni(cod)2.
6.5.2 Nickel vs. Palladium There are a several considerations when choosing an appropriate metal for catalysis which mainly include costs and reactivity. Between nickel and palladium, the former has better economic viability, with costs of 1.20 US$/mol compared to 1500 US$/mol for the latter;341 the main reason for this huge price difference is due to their earth abundance. The estimated abundance of the two transition metals in the earth’s crust is 0.0089 % (nickel) and 6.3 x 10-7 % (palladium),342 which makes the nickel not only cheaper, but also less susceptible to supply fluctuations, a very important consideration in large industrial processes.343
Whilst the previous section showed that most cross-coupling processes were developed using palladium catalysts, some seminal discoveries did involve the use of nickel. Today, an increasing amount of research in nickel catalysis is being conducted not only because of its economic viability, but also because nickel can easily facilitate oxidative addition towards less activated C-LG bonds (like C-OR) whereas in many examples palladium cannot.344–346 Using the Heck reaction as a model, density function theory methods have been applied by Guo and co-workers to show that nickel catalysts trigger a faster oxidative addition than palladium catalysts due to its lower activation energy barrier323 and its more electropositive nature, which allows the loss of electron density at the metal centre more readily.339 This characteristic of nickel has been reiterated in separate computational studies by Smith and Morokuma.347,348
In 1977, the group of Swierczewski was one of the first to show that it was possible to add nickel across a C-O bond, which readily facilitated the oxidative addition of a primary alcohol (173) with various Grignard reagents (174) (Scheme 6-10).349 A later study by Dixon and co-workers also reiterated this phenomenon by successfully coupling aryl and vinyl carbamates 9177) with organomagnesium reagents (178).350
6.5 Organonickel chemistry | 109
Scheme 6-10. First reported studies on the activation and oxidative addition of C-O bonds by Nickel catalysis.
Although limited from natural sources, a wide variety of industrially synthesised halides for use in C-C formation reactions are available. There are many examples where nickel is seen to facilitate the cross-coupling of more these relatively expensive aryl bromides and iodides,351 but its ability to easily activate more inert C-O bonds for coupling reactions opens up a plethora of electrophilic partners. There is an abundance of phenols which are both accessible from nature, as well as from synthetic preparations;341 as the C-O bond in phenols have high energy barriers for oxidative addition, transformations of the hydroxyl group into other more reactive leaving groups is desired. Many such phenol derivatives have been demonstrated to be reactive in nickel-catalysed systems, including, but not limited to, functional groups such as triflates,352 mesylates,353 tosylates,354 sulfamates,355 carbonates,355 carbamates,352 and ethers.356 Generally, phenol derivatives are less costly and greener than their halide counterparts; as well, the ease of functionalisation of phenols for such substrates makes them very attractive starting materials.
Palladium systems have shown to be able to activate some phenol derivatives, but these studies are mainly limited to triflate,357 tosylate358 and mesylate359 substrates; however, these three phenol derivatives are somewhat less desirable than others available. Aryl triflates are fairly unstable, and costly to synthesise, due to the use of triflic anhydride. Aryl tosylates, although quite easy and cheap to make, have high molecular weights which decreases atom efficiency; the use of easily sensitised agent, tosyl chloride, is also another drawback. Highly toxic mesyl chloride is required for the synthesis of mesylates. Extensive reviews of nickel-catalysed C-O bond activation reactions are 110 | Chapter 6: General Introduction - P a r t I I available;326,339,341,360,361 it is not in the scope of this introductory chapter to deliver a comprehensive summary of the literature, but notable discoveries in the field are presented.
6.5.2.1 Nickel-catalysed cross-coupling reactions with aryl ethers and esters A study was published in 2004 by Dankwardt where a Kumada-type coupling between aryl ethers (181) and arylmagnesium reagents (182) was possible in the presence of nickel (Scheme 6-11).362 Chatani more recently made further developments in this field with Suzuki-type couplings between aryl ethers (181) and boronic esters (184) with
Ni(cod)2; this work was able to eliminate the use of organomagnesium reagents, leading to the significant expansion of the substrate scope of these reactions.356 Subsequent studies showed that this was also possible with alkenyl ethers (186).363
Scheme 6-11. Nickel-catalysed Kumada-type and Suzuki cross-coupling reactions with aryl and alkenyl ethers.
Concurrent publications by Garg and Shi in 2008 reported the Suzuki cross-coupling of aryl esters (Scheme 6-12). Using very similar catalytic conditions, the Garg group were able to show that aryl pivalates (187) were most compatible with boronic acids (188), whereas Shi and co-workers reported the coupling between aryl acetates with boroxine (189).364,365 In the same year, Shi again had extended the work to the Negeshi coupling of napththol pivalates (190) with organozinc reagents (191) under fairly mild conditions.366 Recently, Watson published a methodological study involving coupling of 6.5 Organonickel chemistry | 111 aryl styrenes (193) with aryl pivalates (187) under a Ni(cod)2/DPPF catalytic system which allowed for the formation of selectively (E)-1,2-disubstituted olefins (194).367
Scheme 6-12. Nickel-catalysed cross-coupling reactions with aryl pivalates.
6.5.2.2 Nickel-catalysed cross-coupling reactions with aryl sulfamates, carbonates and carbamates Sulfamates, carbamates and carbonates are also easily accessible from phenols, and all three types of C-O bonds have shown to be activated in nickel-catalysed Kumada and Suzuki–Miyaura cross-coupling reactions. Although these phenol-derived functional groups are less atom efficienct than their halide counterparts, they’re more readily accessible and provide a much larger scope of compounds. The year 1989 saw the first report of a carbamate coupling by Kocienski and Dixon where vinyl carbamate (177) was coupled with alkyl Grignard reagents (178) using Ni(acac)2 and (dppe)NiCl2 as catalysts (Scheme 6-10).350 In 1992, Snieckus demonstrated that carbamates can be 352 coupled with Grignard reagents in a mild Ni(acac)2 system. Following this study, the same authors reported a Kumada-type coupling of aryl sulfamates (195) using a Ni- NHC complex, NiCl(Cp)(IMes), for the formation of simple biaryls, N-protected anilines, and azabiaryls (Scheme 6-13).368 112 | Chapter 6: General Introduction - P a r t I I
In 2009, Garg and coworkers published the first successful Suzuki–Miyaura reaction between aryl sulfamates (195) and arylboronic acids (196). In this report, aryl carbamates (197) and carbonates (198) were also successfully activated for coupling in 355 the NiCl2(PCy3)2 based catalytic system. Concurrently, Snieckus et al. also published a similar method to cross-couple aryl carbamates (197) with boronic acids (196) in the 369 presence of additional PCy3HBF4 ligand. (Scheme 6-13) In the same year, Nakamura performed Kumada couplings with aryl carbamates using Ni(acac)2 and hydroxyphosphine.370
Scheme 6-13. Nickel-catalysed cross-coupling reactions with aryl sulfamates, carbamates and carbonates.
6.5.2.3 Nickel-catalysed cross-coupling reactions with aryl alcohols and other inherently inert functional groups In 2010, the first nickel-catalysed aryl alcohol coupling was observed by Shi and co- workers.371 By first treating 2-naphthols (199) with methylmagnesium bromide (200), followed by the desired arylmagnesium bromide (201), C-C bond formation was achieved in the presence of NiF2 and PCy3 (Scheme 6-14). It is proposed that the reaction proceeds via the formation of a magnesium naphtholate complex†††††† which allows the in situ formed nickel(0) complex to add across the C-O bond.
†††††† Structure of magnesium naphtholate complex, 2-NaphOMgBr: 6.6 Project aims | 113
Scheme 6-14. Kumada couplings of 2-naphthols by Shi et al.360
In light of the progress made in nickel-catalysed cross-couplings of aryl alcohols, vinyl and aryl phosphates have also been shown to be activated in Kumada,372,373 Suzuki– Miyaura374,375 and Negishi cross-couplings.376 Other inert functional groups such as aryl fluorides have been shown to be activated to partake in Kumada377,378 and Suzuki– Miyaura379 cross-couplings. Addition into C-N bonds in aryl cyanides have also been observed.380,381
6.6 Project aims The brief summary of the history of C-C cross-coupling and organonickel chemistry have hopefully exposed to the reader that earth-abundant nickel has great potential to interchange expensive palladium in catalysis with further research and development. Apart from its ability to easily facilitate oxidative additions into C-O bonds, limitations of nickel-catalysed systems certainly remain. Some of the issues associated with comparatively higher catalytic-loadings, limited substrate scopes and types of reactions need to be addressed. Furthermore, many of the reaction conditions described previously are quite harsh. As exemplified in the previous sections, the literature is dominated by the use of air-sensitive Ni(cod)2 and (PCy3)2NiCl2. Indeed, these two nickel complexes have numerous characteristics that make them very valuable to the cross-coupling field, but the air-sensitive nature of the prior nickel complex makes its transport difficult, and its preparation is not trivial; the latter nickel source is cheap and easy to prepare, but it is only one of very few nickel(II) systems that do not require additional activation or reducing agents.382–384
At the initiation of this project within the Stewart group, only a very limited amount of air-stable Ni(0) catalysts had been described in the literature. In the past three years, several research groups have reported a variety of such Ni(0) sources.385–388
114 | Chapter 6: General Introduction - P a r t I I
6.6.1 Catalytic activity of air-stable Ni(0) complexes by the Stewart research group To address the matters described above, the Stewart research group initiated an investigation into the development of air-stable Ni(0) sources and have since reported the synthesis of five new air-stable and simply prepared nickel(0) phosphine-phosphite catalysts;389 a succinct discussion of their discovery is presented in chapter 7. These catalysts, along with several homoleptic nickel(0) phosphite complexes were tested for their catalytic activity in Buchwald–Hartwig type amination reactions with aryl and primary alkyl amines as well as in the direct coupling of ammonia.390 Although limitations such as a restricted substrate scope and high catalyst loading also remain for these systems, these catalysts have shown that they are possible alternatives to pre- existing air-sensitive nickel and more expensive palladium reagents.
Prior to the discovery of the new air-stable nickel(0) phosphine-phosphite catalysts, a small investigation of the reactivity of homoleptic Ni[P(OEt)3]4 and Ni[P(OPh)3]4 complexes in the cross-coupling reaction of electron-rich olefins was conducted. This PhD study continues the exploration of the catalytic activity of the new nickel(0) systems in these Heck-Mizoroki reactions. Although the main Heck-Mizoroki investigation reviews the cross-coupling reactions between phenol derivatives, such as aryl triflates and tosylates, and electron-rich olefins, the use of these nickel(0) catalysts for the cross-coupling between aryl bromides and styrenes will be briefly studied for an alternate economical pathway for the synthesis of the previously described antibacterial 135C (52) and its analogues (204) (Scheme 6-15). The suitability of the nickel(0) phosphine-phosphite systems in Suzuki–Miyaura cross-coupling reactions between aryl boronic acids and phenol derivatives will also be investigated.
Scheme 6-15. Alternative synthesis of 135C and analogues through nickel-catalysed Heck-Mizoroki cross-coupling reactions.
6 .6 Project aims | 115
6.6.2 Summary of project aims There are two primary aims in this part of the thesis: i. The evaluation of the catalytic activity and scope of the newly synthesised Ni(0) complexes in the Mizoroki–Heck and Suzuki–Miyaura cross-coupling reactions, and; ii. Explore the possibility of an alternative synthesis of compound 135C and its analogues by utilising nickel catalysis instead of palladium.
116 | Chapter 7: Mizoroki – Heck Cross - Coupling Reactions
Chapter 7: Mizoroki–Heck Cross-Coupling Reactions 7.1 Mizoroki–Heck reaction of electron-rich olefins with nickel(0) phosphites Systems involving stable Ni(II) sources can be used for some cross-coupling reactions, however, these reactions often require additional reagents to reveal the active nickel species;391,392 Ni(0) systems have also been reported but many such catalytic processes use the highly air-sensitive, and relatively expensive reagent Ni(cod)2 as the Ni(0) source.333 At the beginning of this study, it was envisioned that nickel phosphite complexes would be able to replace palladium catalysts in the Mizoroki–Heck reaction between electron-rich olefins with aryl halides. Previous studies by Skrydstrup et al. have shown that a reaction mixture including Ni(cod)2, and the bidente ligand DPPF can cross-couple aryl triflates (205) with butyl vinyl ether (206) (Scheme 7-1).393 This reaction provided the 1,1-disubstituted olefin product (207), and as this olefin is hydrolytically unstable, treatment with aqueous HCl provided the investigators with the corresponding ketone (208) for easy isolation in high yields.
The investigation in our research group compared the reactivity of Ni(cod)2 with that of
Ni[P(OPh)3]4 and Ni[P(OEt)3]4, nickel phosphites that are easily prepared from hexahydrate forms of nickel nitrate and nickel chloride, respectively.394,395 An optimisation study was performed, and the results showed that, for the reaction between phenyl p-tosylate and butyl vinylether, the reactivity of Ni[P(OEt)3]4 is comparable to the Skrydstrup benchmark reaction (78 %) with a yield of 85% (Scheme 7-1). A scope of this reaction was carried out, however, it was established that only a limited range of substrates were tolerated, and further studies were required to improve on its efficiency and substrate tolerance.
Reagents and conditions: Skrydstrup 2011- Ni(cod)2 (5 mol%), DPPF (5 mol%), Cy2NMe (3.0 equiv.), 1,4-dioxane, 100 °C, 78 %; Stewart 2015- Ni[P(OEt)3]2 (2 mol%), DPPE (2 mol%), Cy2NMe (1.0 equiv.), toluene, 100 °C, 85 %. Scheme 7-1. Benchmark Mizoroki–Heck reaction using air-sensitive Ni(cod)2 by 393 390 Skrydrup et al. and air-stable Ni[P(OEt)3]4 with by Stewart et al.
7.1 Mizoroki – Heck reaction of electron - rich olefins w i t h nickel(0) phosphites | 117
Previously, phosphine-phosphite catalytic systems have been reported to be used in 396 397 combination with Ni(cod)2, and other nickel precatalysts, as well as with rhodium398–400 and copper401–403 complexes. During the subsequent investigations into using nickel phosphites within our group, it was discovered that a new air-stable nickel(0) phosphine-phosphite complex, (dppf)Ni[P(OPh)3]2 (209) was suitable for a 404 range of C-N cross-coupling reactions. This (dppf)Ni[P(OPh)3]2 phosphine-phosphite complex could be prepared from a simple procedure of stirring Ni[P(OPh)3]4 (210) with DPPF in toluene at 100 °C. Interestingly, the reaction was shown not to form any of the
Ni(dppf)2, and was high yielding at 93 % (Scheme 7-2).
Reagents and conditions: i) P(OPh)3 (4.0 equiv.), NaBH4, EtOH, -60 °C RT, 15 min; ii) DPPF (2.0 equiv), toluene, reflux, 2 h Scheme 7-2. Synthesis of new air-stable nickel(0) complex, (dppf)Ni[P(OPh)3]2.
Following these promising results, the syntheses of other easily accessible nickel(0) catalysts were conducted within the group, which led to a series of eight nickel(0) complexes. These include three simple homoleptic complexes Ni[P(O-p-tol)3]4, Ni[P(O- m-tol)3]4 and, Ni[P(O-o-tol)3]3, four new phosphine-phosphite nickel(0) complexes (211-214, Figure 7-1), and a chelate diphosphate nickel(0) complex (215, Scheme 7- 389 2). The prior four complexes were prepared in a similar way as (dppf)Ni[P(OPh)3]2, beginning with the synthesis of the corresponding phosphite tetrakis, which can then be refluxed with the appropriate phosphine ligand for the desired complexes.
118 | Chapter 7: Mizoroki – Heck Cross - Coupling Reactions
Figure 7-1. Four new phosphine-phosphite nickel(0) complexes. The chelate diphosphate nickel(0) complex (215) was prepared differently from the other four complexes. The chelating phosphite 216 was first prepared in 83 % yield by treating 2,2’-biphenol (217) with phosphorous trichloride and triethylamine. Following isolation, this phosphite (216) was reacted with Ni(cod)2 for the diphosphate nickel(0) complex Ni(BiPhenOPhos)2 (215) (Scheme 7-3).
Reagents and conditions: i) PCl3, NEt3, THF, -40 °C → RT, 2 h; ii) Ni(cod)2, toluene, 100 °C, 1 h. Scheme 7-3. Synthesis of the chelate diphosphite nickel(0) complex, Ni(BiPhenOPhos)2).
With these new phosphine-phosphite catalysts in hand, the Mizoroki–Heck reaction was revisited to determine whether an improvement in yield or scope was possible.
7.2 Mechanism of the Mizoroki–Heck cross-coupling reaction As previously stated in the introductory chapter, the Mizoroki–Heck cross-coupling reaction is now one of the most widely-used synthetic routes for carbon-carbon bond formations.116,321 The mechanism of the Mizoroki–Heck reaction follows the general transition-metal mediated catalytic cycle as outlined in Figure 6-5, but is mechanistically different to other cross-coupling reactions in that it does not contain a transmetallation step.324 Rather, the two reacting partners are coupled through the olefin coordination, and the C-C bond formation occurs through the migratory insertion (of the olefin) and not during a reductive elimination step. A more thorough discussion is presented in this section, using the model reaction between phenyl halide and butyl vinyl ether as an example (Scheme 7-4).
7.2 Mechanism of the Mizoroki – Heck cross - c o u p l i n g r e a c t i o n | 119
Scheme 7-4. The proposed catalytic cycle of the Mizoroki–Heck cross-coupling reaction between phenyl p-tosylate and butyl vinylether.
0 After the in situ generation of the catalytically active species, M L2, the oxidative 0 addition of the coupling partner, usually an aryl or vinyl halide (218), with the M L2 readily forms a metal(II) complex, L2MRX (219) (step A). The metal centre of this metal(II) complex can then further coordinate with the olefin (206) (step B) in preparation for a migratory syn-insertion (step C).115,116 At this point, it is imperative to have a closer look at a few mechanistic considerations that determines the regioselectivity of the olefin and the mechanistic pathway of the insertion.
Depending on the nature of the oxidative addition product, the insertion mechanism of the catalytic cycle proceeds differently, but delivers the same cross-coupled product. Cationic OA products undergo the dissociation of a solvent molecule, whereas neutral OA products undergo the dissociation of a phosphine molecule (Scheme 7-5, polar(cationic)/neutral pathway, respectively). Palladium species that are cationic in nature are usually formed from the oxidative addition of triflates and diazonium salts, 120 | Chapter 7: Mizoroki – Heck Cross - Coupling Reactions whereas neutral species are formed from halides.405 The prior cationic palladium species are also more reactive towards π-complexation of alkenes than their neutral counterparts, which allows for milder conditions used in such Mizoroki–Heck reactions.393,406
Scheme 7-5. Neutral and polar pathways of the olefin coordination.
The regioselectivity of the Mizoroki–Heck reaction can result in either a 1,2 or a 1,1- disubstituted product; the two pathways leading to these products are exemplified in Scheme 7-6. The orientation of the coordinating olefin can lead to two different disubstituted products, either a linear 1,2- (220) or a branched 1,1- product (221).116 The regiochemistry is dependent on both the steric and electronic nature of the coupling olefin. Electron-withdrawing olefins usually favour 1,2-linear products (220), and electron-donating olefins generally yield 1,1-branched products (221).290,405 In the discussed study with electron-rich butyl vinylether, the formation of 1,1-disubstituted product (222) is expected.
Scheme 7-6. Regioselectivity dependant on the olefin insertion.
After the C-C bond is formed, internal rotation ensues to give an arrangement of a β- hydrogen and the metal on the same face (step D). Not only does this syn-periplanar 7.3 Optimisation of the nickel phosphite catalysed M i z o r o k i – Heck reaction | 121 orientation allow the elimination process to occur, this also determines the E/Z configuration of the product (222) (step E).116 The remaining metal hydride complex,
L2MHX (223), is then reduced by a base for the regeneration of the M(0) catalyst (step F), which is turned over in the catalytic cycle.
7.3 Optimisation of the nickel phosphite catalysed Mizoroki–Heck reaction Preliminary reactions between 4-cyanotriflate (205) and vinylbutylether (206), using the
(binap)Ni[P(OPh)3]2 (214), were conducted in the absence and presence of the BINAP, resulting in none of the desired product 207 (Table 7-1, entry 1&3). The catalyst
(dppf)Ni[P(OPh)3]2 was also trialled and only resulted in a GC conversion of 3 % to 207 (Table 7-1, entry 2). The series of eight nickel(0) complexes were also trialled with an appropriate amount of DPPF as the ligand, and unfortunately, little conversion was observed. The tetrakis, and m- and p-tolyl nickel(0) phosphites did not catalyse the reaction, similarly, the nickel DPPF/phosphite 209 also failed (Table 7-1, entries 4-7).
The Ni[P(O-o-tol)3]3 gave a conversion of only 1 % (Table 7-1, entry 9). DPPF as an additive to (dppf)Ni[P(OPh)3]2 resulted in a conversion of 8 % (Table 7-1, entry 8), whereas Ni(BiPhenOPhos)2 allowed for a 24 % GC-conversion and 31.5 % isolated yield (Table 7-1, entry 10). Unfortunately, when the latter reaction was repeated, no conversion was observed, therefore, we then reverted back to using (dppf)Ni[P(OPh)3]2 to continue our investigations.
122 | Chapter 7: Mizoroki – Heck Cross - Coupling Reactions
Table 7-1. Screening of the new nickel(0) complexes in the Mizoroki–Heck cross- coulpling reaction between 205 and 206.
Conversion Entry Cataltyst (mol%) Ligand (mol%) (%)[b] 1 (binap)Ni[P(OPh)3]2 (5) - n.r. 2 (dppf)Ni[P(OPh)3]2 (5) - 3 3 (binap)Ni[P(OPh)3]2 (5) BINAP (5) 1 4 (dppf)Ni[P(O-m-tol)3]2 (5) DPPF (5) 0 5 Ni[P(O-m-tol)3]4 (5) DPPF (10) 0 6 Ni[P(O-p-tol)3]4 (5) DPPF (10) 0 7 (dppf)Ni[P(O-p-tol)3]2 (5) DPPF (5) 0 8 (dppf)Ni[P(OPh)3]2 (5) DPPF (5) 8 9 Ni[P(O-o-tol)3]3 (5) DPPF (10) 1 10 Ni(BiPhenOPhos)2 (5) DPPF (10) 24 (31.5)* [a] All reactions were conducted between a 0.3-0.5 mmol scale, using 4.0 equiv. butyl vinyl ether [b] and 3.0 equiv. Cy2NMe. Determined by GC. Isolated yields are reported in brackets. *not repeatable.
In all the GC spectra collected up to this point, the presence of 1-(1-isobutoxyethoxy)-2- methylpropane (224) had been detected; this compound was presumed to be the product of a side reaction with two molecules of vinylbutylether.‡‡‡‡‡‡ In an attempt to achieve a higher conversion, we lowered the equivalents of alkene from 4 to 1.5 and base from 3 to 1. This change increased the GC conversion dramatically to 45 %, and a 39 % yield was isolated for (dppf)Ni[P(OPh)3]2. With these improved conditions, a range of organic and inorganic bases were trialled. It was also noted that the di-ether product 224 was still present in small amounts.
A series of different bases were tested but the yield was not improved upon the initially obtained 39 %. Triethylamine and Hünig’s base (DIPEA) gave similar conversions and yields of 10 % (12 %) and 11 % (9 %), respectively (Table 7-2, entries 3-4), whereas other organic bases (sodium tert-butoxide and diisoproylamine) were ineffective. Sodium, potassium and cesium carbonates were also trialled, with the former two giving
‡‡‡‡‡‡1-(1-isobutoxyethoxy)-2-methylpropane (224)
7.3 Optimisation of the nickel phosphite catalysed M i z o r o k i – Heck reaction | 123 conversions of 3 and 4 % (Table 7-2, entries 6-7), respectively. Cesium carbonate in the presence and absence of the phase-transfer catalyst tetra-n-butylammonium bromide (TBAB) under the standard conditions did not allow the reaction to proceed (Table 7-2, entries 8-9).
Table 7-2. Base screening for the (dppf)Ni[P(OPh)3]2 – catalysed cross-coupling between 205 and 206.
Entry Base (equiv.) Conversion (%)[b] 1 Cy2NMe (1) 45 (39) 2 NaOtBu (1) 0 3 Et3N (1) 10 (12) 4 DIPEA (1) 11 (9) i 5 Pr2NH (1) 0 6 Na2CO3 (1) 3 7 K2CO3 (1) 4 8 Cs2CO3 (1)/TBAB (1) 0 9 Cs2CO3 (1) 0 [a] All reactions were conducted between a 0.3-0.5 mmol scale, using 1.5 equiv. butyl vinyl ether. [b] Determined by GC. Isolated yields are reported in brackets.
Subsequently, the role of the ligand partner in the reaction was investigated. The similarly bridged 1,4-bis(diphenylphosphino) ethane (DPPE) and butane (DPPB) ligands gave negligible yields (Table 7-3, entries 1 & 8), similar to the bidentate and bulky XantPhos and DavePhos ligands (Table 7-3, entries 6-7). Interestingly, the BINAP ligand was compatible in the reaction, giving a 64 % conversion and 42 % isolated yield (Table 7-3, entry 2). Of the three commercially-available and more sterically demanding DPPF-derived ligands (Figure 7-2), the isopropyl analogue gave the best yield at 65 % (Table 7-3, entry 5), whereas the tert-butyl and cyclo-hexyl analogues produced lower yields of 25 % and 20 %, respectively (Table 7-3, entries 3- 4). 124 | Chapter 7: Mizoroki – Heck Cross - Coupling Reactions
Figure 7-2. Structures of 1,1'-Bis(diphenylphosphino)ferrocene (DPPF) and its tert- butyl (DtBuPF), cyclohexyl (DCyPF), and isopropyl (DiPrPF) analogues.
Table 7-3. Ligand screening for the (dppf)Ni[P(OPh)3]2 – catalysed cross-coupling between 205 and 206.
Entry Ligand (mol%) Conversion (%)[b] Yield (%) 1 DPPE (5) 1 0 2 BINAP (5) 64 42 3 DtBuPF (5) 41 25 4 DCyPF (5) 34 20 5 DiPrPF (5) 59 65 6 XantPhos (5) - 3 7 DavePhos (5) - 0 8 DPPB (5) - 0.8 t 9 Bu3PHBF4 (10) 41 29 [a] All reactions were conducted between a 0.3-0.5 mmol scale, using 1.5 equiv. butyl vinyl ether. [b] Determined by GC.
Following this process, the role of the ratio of reagents and reaction time was investigated. When the reaction time was increased to 48 hours, the product yield dramatically decreased to 24 % (Table 7-4, entry 1), and surprisingly, as does increasing the catalyst and ligand loading to 10 mol% (Table 7-4, entry 2). Decreasing and increasing the ligand to catalyst ratio diminishes the yield to 32 % and 57 %, respectively (Table 7-4, entry 3). Subsequently, the optimised conditions of this reaction at this stage of the project remained to be those outlined in Table 7-3, entry 5.
7.3 Optimisation of the nickel phosphite catalysed M i z o r o k i – Heck reaction | 125
Table 7-4. Ratio of reagents in the cross-coupling between 205 and 206.
i Entry Ni[P(OPh)3]2(dppf) D PrPF Yield (%) 1 5 mol% 5 mol% 24 [c] 2 10 mol% 10 mol% 35 3 5 mol% 2.5 mol% 32 4 5 mol% 10 mol% 57 [a] [b] All reactions were conducted between a 0.4 mmol scale, using 1.5 equiv. butyl vinyl ether. Isolated yield. [c] Reaction was run for 48 hours.
Although the optimised conditions from this set of nickel(0) complexes was not as high yielding as the reaction catalysed by Ni[P(OEt)3]2 (65 % and 85 %, respectively), an exploration into the substrate scope was performed to investigate whether the
(dppf)Ni[P(OPh)3]2 system would tolerate a broader range of aryl triflate and halide coupling partners.
7.3.1 Nickel phosphite catalysed Mizoroki–Heck reaction of 4-chlorobenzonitrile 225 It was hoped that the reaction would be able to proceed with 4-chlorobenzonitrile, as this reagent is inexpensive and commercially available, whereas 4-cyanophenyltriflate (205) requires preparation using expensive triflic anhydride. A short investigation on the Mizoroki–Heck cross-coupling reaction between 4-benzonitrile and vinyl butylether was carried out.
Scheme 7-7. Mizoroki–Heck cross-coupling reaction between 225 and 206.
The optimised conditions from section 7.3 using (dppf)Ni[P(OPh)3]2 and DiPrPF resulted in a dismal conversion of 2 %. The use of other 1,1’-disubstituted phosphino ferrocene ligands (Figure 7-2) and an increase in catalyst and ligand loading to 10 mol% 126 | Chapter 7: Mizoroki – Heck Cross - Coupling Reactions were also unsuccessful in producing the desired compound 207. The same ligand screening, with the addition of BINAP, was carried out with Ni[P(OPh)3]4 as the catalyst. Conversions of 7 and 2 % were achieved with BINAP and DtBuPF, respectively, and the ligands DtBuPF, DCyPF, and DiPrPF were even less compatible in the reaction system with no conversion to the acetophenone 207 detected.
Within the group, a previously optimised Ni[P(OEt)3]4 condition for the reaction 225 to
207 (Scheme 7-7) gave only a GC conversion of 7%. A screening of Ni[P(OEt)3]2 with BINAP, DPPF, and the three DPPF analogues (Figure 7-2) as the ligand partner showed an improvement in conversion to 19% with the cyclohexyl derivative (DCyPF). Surprising, the similarly structured DPPF, DtBuPF and DiPrPF ligand systems showed no reaction, neither did the BINAP ligand. As the catalytic activity of the trialled nickel(0) phosphites was not well received by the cheaper electrophilic coupling partner, this investigation was not further pursued.
7.4 Scope of the nickel phosphite catalysed Mizoroki–Heck reaction 7.4.1 Reactivity of phenylhalides and pseudohalides With the optimised conditions in hand from section 7.3, a substrate scope was explored next. In the first instance, phenyl halides and pseudohalides were tested to compare the reactivity of the C-X bond in the reaction outlined in Scheme 7-8. The three phenylhalides and phenyltosylate were not reactive and showed no conversion, whereas phenyltriflate resulted in only a 19 % conversion based on GC-MS.
Scheme 7-8. Mizoroki–Heck reaction between phenyl halides/pseudohalides and 206.
Comparing these results to the reactions presented in section 7.3.1 with 4- chlorobenzonitrile (225), it was observed that a strong electronically deactivating substituent was required for minimal activity. Additionally, it was quite surprising that the less electronegative bromide and iodide coupling partners were unsuccessful in this reaction, given their reactivity profile in other cross-coupling reactions. Interestingly, 7.4 Scope of the nickel phosphite catalysed Mizoroki – H e c k r e a c t i o n | 127 large amounts of starting material in the phenyl tosylate and triflate reactions were detected; however, all three phenyl halides were consumed in the reaction with no product production, however no homocoupled product was detected. A subsequent reaction using 4-cyanophenyltosylate was performed to determine whether an electron withdrawing substituent would have a positive effect on the phenyl tosylate coupling partner, as it does on the corresponding triflate; gratifyingly, a quantitative conversion was observed and a high 73 % yield was isolated. This result is very promising for the initiation of a further substrate screen. However, to fully understand the catalytic cycle, a thorough mechanistic investigation would be required.
7.4.2 Scope of the aryl triflate coupling partner with butyl vinylether Twelve substrates were chosen to carry out the substrate screening, only four candidates showed any conversion into their corresponding desired products. Three of these reactions were fully isolated under standard workup conditions. Weakly activating electron-donating substituents rendered the phenyltriflate entirely unreactive with the catalyst system, and only starting material was identified in the reaction mixture without any desired products (227, 228 and 229). 1- (230) and 2- (231) naphthalene triflates produced high conversions and isolated yields of 56 % (61 %) and 77 % (100 %), respectively; a similarly structured but slightly more electron-donating 6-quinoline triflate rendered the reaction unreactive (232). From the trends observed thus far, electron-donating substituents were detrimental to this catalytic system, with the methoxy (233 and 234) and acetamide (235) functionalised candidates following suit. However, ethyl ester 236 presented a GC conversion of 10 %. At the other end of the spectrum, strongly electron-withdrawing 4-nitro phenyltriflate was unsuccessful (237) in the reaction. 3-Trifluoromethyl phenyltriflate was consumed in the reaction, but no product was isolated (238).
128 | Chapter 7: Mizoroki – Heck Cross - Coupling Reactions
Table 7-5. Scope of the aryl triflate coupling partner 239.
[a] All reactions were conducted on a 0.26-0.5 mmol scale using 1.5 equivalents of vinyl butyl ether (206). GC conversions are indicated in brackets (GC).
Unfortunately, a large range of functional groups were not well tolerated in this cross- coupling reaction catalysed by the newly developed air-stable nickel(0) complex,
(dppf)Ni[P(OPh)3]2. The earlier established Ni[P(OEt)3]4 catalysed reactions were reported in a higher yield (85 %) than those obtained in this investigation (65 %). Although the yield of the model reaction presented by Skrydstup was slightly lower (78
%) than that of the Ni[P(OEt)3]4-catalysed counterpart, Ni(cod)2, with its undesirable instability, currently still appears to be the superior nickel catalyst for Mizoroki–Heck reactions with electron-rich olefins (Scheme 7-1).
7.4.2 Application of nickel(0) phosphite system on the synthesis of compound 135C As presented in Part I of this thesis, the key C-C bond formations of the novel antibacterial agent 135C and its analogues were synthesised through the use of a tandem palladium-catalysed Mizoroki–Heck reaction. To investigate whether nickel complexes could replace the palladium catalyst in this reaction, the reaction between 1,3,5- 7 . 4 S cope of the nickel phosphite catalysed Mizoroki – H e c k r e a c t i o n | 129 tribromobenzene (53) and styrene 54 was carried out using the
(dppf)Ni[P(OPh)3]2/DiPrPF system. Unfortunately, the coupling partners were not tolerated in this reaction. Similar to the investigation of the nickel-catalysed reactions of phenyl halides (Section 7.4.1), it appears that the aryl halide was consumed in the reaction but without the presence of the desired product.
Scheme 7-9. Unsuccessful synthesis pathway to novel antibacterial compound 135C.
130 | Chapter 8: Suzuki – Miyaura Cross - Coupling Reactions
Chapter 8: Suzuki–Miyaura Cross-Coupling Reactions 8.1 Suzuki cross-coupling reactions In 1979, the first reports on the palladium-catalysed cross-coupling reaction between organoboron compounds (241) with vinyl and aryl halides (242) were published by Suzuki, Miyaura and Yamada (Scheme 8-1).301,302 Subsequent development of this C-C forming method has seen its popularity rise in the fine chemical and pharmaceutical industries because of its use of organoboron compounds. Organoboron compounds have a broad functional group tolerance, chemically stable, and are their corresponding reactions can be highly chemoselective. Moreover, from a practical point of view, they are non-toxic, inexpensive and are easily prepared.407 With the added incentive that these reactions can be run under very mild conditions, the Suzuki–Miyaura reaction developed to be one of the most used C-C bond formation methods in chemistry.408
Scheme 8-1. General pattern of the palladium or nickel catalysed Suzuki–Miyaura cross-coupling reaction.
To this date, palladium-catalysed Suzuki–Miyaura reactions still dominate the literature. That said, nickel catalysts have been used as a replacement for the palladium counterpart in the Suzuki–Miyaura cross-coupling reaction for some years, with one of the first reports by Miyaura et al. on the cross-coupling of chloroarenes with aryl boronic acids in 1997.409 In the next few years, a number of other reports of nickel- catalysed Suzuki–Miyaura reactions emerged in a short time period, which brought along the development of this earth-abundant metal’s usefulness in the synthesis of C-C bonds between R-boronic acids or esters with appropriate coupling partners containing leaving groups.409–414 The switch from palladium to nickel has grown in popularity since these first reports, and recent advances in the topic has seen may new systems which can be applied in these cross-couplings.415–417
This chapter deals with the investigation of air-stable nickel(0) catalysts as alternatives to the currently used nickel and palladium counterparts for their application in the Suzuki–Miyaura cross-coupling reactions. 8.2 Mechanism of the S u z u k i – Miyaura cross - c o u p l i n g r e a c t i o n . | 131
8.2 Mechanism of the Suzuki–Miyaura cross-coupling reaction. The mechanism of the Suzuki–Miyaura reaction follows the generic three-stage process of oxidative addition-transmetallation-reductive elimination sequence, as described previously in figure 6-5. The first step is the same as in the Mizoroki–Heck cross- coupling mechanism (Scheme 8-2), where after the in situ generation of the catalytically 0 active species, M Ln, oxidative addition of the metal into the carbon-halide takes place for the formation of metal(II) complex, L2MRX (219) (step A).
Scheme 8-2. Catalytic cycle of the Suzuki–Miyaura cross-coupling reaction between boronic acids and halides/pseudohalides.
At this point, the pathway digresses from the Mizoroki–Heck mechanism. Early observations by Suzuki and Miyaura proposed that, at this stage, the metal(II) complex,
L2MRX (219) undergoes association with four-coordinate “active” boronate species (243) for the formation of the transmetallation product 244 (Scheme 8-3, pathway A).301,302 In the following six years (1979 – 1985), further experimental investigations by the same authors into this mechanistic pathway led to the conclusion that an oxo- palladium pathway (Scheme 8-3, pathway B) is more likely to be the mechanism.418,419 Further computational and kinetic studies by Amatore and Jutand,420–422 Hartwig,423 and Schmidt424 provided compelling evident for the existence of the oxo-palladium pathway. There has only been one study by Matos and Soderquist which gave little 132 | Chapter 8: Suzuki – Miyaura Cross - Coupling Reactions support for the catalytic journey through the boronate pathway A.425 Hence, the ligand exchange step is now believed to be next in the catalytic cycle, where the selected base anion displaces the halide or pseudohalide moiety from the oxidative addition product II R-M-X (219), to give a more reactive species, R1-M -b (245) (step B); a variety of bases have been used in Suzuki–Miyaura reaction, which allows the formation of more reactive hydroxides, alkoxides and phosphates.355,358,426
Scheme 8-3. The boronate (A) and the oxo-palladium (B) pathways of the transmetallation step in Suzuki–Miyaura reaction as proposed by the authors.
As discussed immediately above, the intermediate 246 is proposed to be formed via the oxo-metal pathway which then undergoes a transmetallation step with a boronate complex (247) to form the organometal species 244 (step D); the boronate complex is activated by a second equivalent of base (step C). This step in the catalytic cycle, as well as the base/ligand exchange, exemplifies the importance of the role of the base in the reaction and distinguishes the Suzuki–Miyaura reaction from most other cross- coupling reactions. Reductive elimination (step E) of the metal complex 244 releases the C-C coupled product 248 and regenerates the catalyst for the continuation of the catalytic cycle.
8.3 Suzuki–Miyaura cross-coupling reaction of aryl pseudohalides and boronic acid. As part of the investigations into the applicability of the series of nickel(0) phosphite catalysts, previous group work by Sven Kampmann had embarked on a preliminary investigation into their reactivity in the Suzuki reaction. In this trial, the potential of
(dppf)Ni[P(OPh)3]2 was examined. For the biphenyl product 249, the reaction between phenylboronic acid (250) and 4-cyanophenyl triflate (205) produced a yield of 21 %, 8.4 Optimisation Studies | 133 whereas when coupled with 4-cyanophenyl tosylate (251), the yield was 31 %. In comparison, Monteiro’s Suzuki reaction between 250 and 251 catalysed by the also air- 417 stable NiCl2(PCy3)2, produced a near quantitative yield of 96 %. It was in the scope of this current proposed investigation to optimise our nickel catalytic system to hopefully produce comparable yields and to contribute to the nickel(0) catalysis literature.
Scheme 8-4. Preliminary reactions between aryl pseudohalides (205 and 251) and phenylboronic acid (250) using the (dppf)Ni[P(OPh)3]2/DPPF system.
8.4 Optimisation Studies§§§§§§ Initially, ten nickel (0) phosphine-phosphite complexes from the Mizoroki–Heck investigation were screened for the model coupling reaction between 4- cyanophenyltosylate (251) and phenylboronic acid (250), in an attempt to increase upon the preliminary screened isolated yield of 31 %. The investigation began with
(binap)Ni[P(OPh)3]2 with BINAP as an additonal ligand, providing a near quantitative consumption of starting material but a yield of only 22 % (Table 8-1, entry 1). Although this catalytic system provided a low yield, it confirmed that such nickel(0) phosphine- phospite system can indeed be applied successfully to Suzuki–Miyaura cross-coupling reactions. Following this promising result, the other nine nickel(0) complexes were trialled with DPPF as the partner ligand. In each of these trails, the loading of the amount of ligand used corresponded to the loading of the catalyst: equal amount for the heteroleptic complexes, twice the amount for the homoleptic catalysts. The amount of ligand is likely to have an effect on the ligand dissociation equilibrium which provides the catalytically active nickel species at the beginning of the catalytic cycle. In the previous studies on the Mizoroki–Heck cross-coupling and C-N cross-coupling reactions utilising these nickel phosphites, a 1:2 ratio between the homoleptic nickel
§§§§§§ In this optimisation study, GC conversions (area percent quantitation) were used as an indication of the consumption of starting material, and as an estimate of the amounts of analytes present. GC yields (multiple point internal standard method) were obtained using docosane as an internal standard; this method accounts for variances in relative response factors of the GC system to the analytes. 134 | Chapter 8: Suzuki – Miyaura Cross - Coupling Reactions
species and diphosphine ligands is favourable to the catalytic process and yields, over a 1:1 ratio.389
Table 8-1. Testing of various nickel(0) complexes in the Suzuki cross-coupling reaction between 4-cyanophenyltosylate (251) and phenylboronic acid (250).
Conversion Yield Entry Catalyst (mol%) Ligand (mol%) (%)[b] (%)[b] 1 (binap)Ni[P(OPh)3]2 (5) BINAP (5) 98 22 2 Ni[P(OEt)3]4 (5) DPPF (10) 0 0 3 (dppf)Ni[P(O-m-tol)3]2 (10) DPPF (10) 100 57 4 Ni[P(O-m-tol)3]4 (5) DPPF (10) 100 56 5 (dppf)Ni[P(O-p-tol)3]2 (5) DPPF (5) 100 55 6 Ni[P(O-p-tol)3]4 (5) DPPF (10) 100 49 7 (XantPhos)Ni[P(OPh)3]2 (5) DPPF (5) 100 49 8 Ni(BiPhenOPhos)2 (5) DPPF (10) 99 15 9 Ni[P(OPh)3]4 (5) DPPF (10) 99 37 10 (dppf)Ni[P(OPh)3]2 (5) DPPF (5) 100 45 [a] [b] All reactions were conducted on a 0.2 mmol scale, using 1.0 equiv. phenylboronic acid. Determined by GC with docosane as internal standard.
Out of the five homoleptic catalytic conditions, only the ethyl phosphite derivative,
Ni[P(OEt)3]4, (Table 8-1, entry 2) proved unsuccessful; the other four candidates showed near full conversion of the start material. However, the GC yields of the
products of these reactions varied. The structurally comparable meta-(Ni[P(O-m-tol)3]4)
and para-tolulphosphite (Ni[P(O-p-tol)3]4) catalysts gave similar yields of 56 % (Table 8-1, entry 4) and 49 % (Table 8-1, entry 6), respectively. The difference in yield could be used be due to the slightly more sterically crowded phosphite groups of the Ni[P(O-
m-tol)3]4 complex over its para- counterparts, as more sterically hindered ligands undergo strain-releasing reductive elimination processes faster than their less sterically demanding counterparts.427 Following the trend, the non-substituted triphenylphosphite
catalyst, Ni[P(OPh)3]4 (Table 8-1, entry 9), only produced a yield of 37 %; the absence of the electron donating alkyl groups may also have contributed to the lower yield. The
Ni(BiPhenOPhos)2 complex produced a much lower yield of 15 %, which may be due to its highly stable and rigid structure that perhaps is not able to fully facilitate ligand 8.4 Optimisation Studies | 135 exchange with DPPF for the formation of a catalytically active species (Table 8-1, entry 8).428
Of the heteroleptic complexes, all four candidates assisted in the full consumption of the starting material and produced comparable yields between 45-70 %. The
(XantPhos)Ni[P(OPh)3]2 complex produced a slightly higher yield of 49 % (Table 8-1, entry 7) as opposed to (dppf)Ni[P(OPh)3]2 at 45 % (Table 8-1, entry 10). Mistakenly, the (dppf)Ni[P(O-m-tol)3]2 catalyst was loaded at 10 %, however, instead of an expected increase in yield than that of the para- candidate, the values were comparable at 57 % (Table 8-1, entry 3) and 55 % (Table 8-1, entry 5), respectively. Although the conditions described in entry 5 did not produce the highest yield, it had the most efficient catalyst and ligand loading, as well as a comparable yield. Hence,
(dppf)Ni[P(O-p-tol)3]2 was chosen to continue with further optimisation.
Seven different ligands were trialled to determine compatible systems with the air stable
(dppf)Ni[P(O-p-tol)3]2, their structures are as shown in Figure 8-1. It is interesting to note that, although the BINAP ligand provided a 98 % conversion (albeit, low 22 % yield) with the (binap)Ni[P(OPh)3]2 complex, it rendered the process unreactive when in use with (dppf)Ni[P(O-p-tol)3]2 (Table 8-2, entry 2). Structurally, it has the smallest bite angle of all the ligands trialled,429 a factor that may have had negative impacts on the reductive elimination step of the catalytic cycle.
Figure 8-1. Structure of ligands trialled in (dppf)Ni[P(O-p-tol)3]2- catalysed Suzuki cross-coupling reaction between 4-cyanotosylate (251) and phenylboronic acid (250).
136 | Chapter 8: Suzuki – Miyaura Cross - Coupling Reactions
Table 8-2. Ligand screening for the (dppf)Ni[P(O-p-tol)3]2 catalysed Suzuki cross- coupling reaction between 4-cyanotosylate (251) and phenylboronic acid (250).
Entry Ligand (mol%) Conversion (%)[b] Yield (%)[b] 1 DPPF (5) 100 55 2 BINAP (5) 0 0 3 DtBuPF (5) 100 57 4 DPPB (5) 100 70 5 XantPhos (5) 100 52 t 6 ( Bu)3PHBF4 (10) 100 49 7 DiPrPF (5) 100 49 [a] [b] All reactions were conducted on a 0.2 mmol scale, using 1.0 equiv. phenylboronic acid. Determined by GC with docosane as internal standard.
The other five ligands allowed for the entire consumption of starting material and provided comparable yields to the benchmark of 55 % (Table 8-2, entry 1). Similarly structured but branched DtBuPF ligand (Table 8-2, entry 3) produced a slightly higher yield at 57 %, whereas the isopropyl derivative, DiPrPF (Table 8-2, entry 7), produced 49 %. The wide bite angled but more rigid XantPhos generated a comparable yield of 52 % (Table 8-2, entry 5). Versatile cross-coupling conditions as described by Gregory 430 t Fu, utilising the costly tri-tert-butylphosphonium tetrafluoroborate (( Bu)3PHBF4) was also investigated. Although, there was no improvement to the yield at 49 % (Table 8-2, entry 6), we have shown that these conditions can be successful in Suzuki–Miyaura systems. Surprisingly, the very flexible and small 1,4-bis(diphenylphosphino)butane (DPPB) ligand was also very compatible in the system, producing a good yield (70 %) (Table 8-2, entry 4). At $5.04/mmol, DPPB is cost effective as well.
Given the improvement in yield of (dppf)Ni[P(O-p-tol)3]2 combined with DPPB, a short examination into the role of the base was performed (Table 8-3). Organic amine bases, triethylamine (Table 8-3, entry 1), Hünig’s base (Table 8-3, entry 2), and N,N- dicyclohexylmethylamine (Table 8-3, entry 3) had significant deleterious effects on the yield (18, 32, 26 %, respectively). Inorganic base Na2CO3 produced a moderate yield of
58 % (Table 8-3, entry 4), however, the switch to K2CO3 and Cs2CO3 dramatically reduced the yields to 21 and 10 %, respectively. Given these results, the originally 431,432 trialled K3PO4 still stands as the benchmark (Table 8-3, entry 7). 8.4 Optimisation S t u d i e s | 137
Table 8-3. Base screening for the Ni[P(O-p-tol)3]2(dppf) catalysed Suzuki cross- coupling reaction between 4-cyanotosylate (251) and phenylboronic acid (250).
Entry Base (equiv.) Conversion (%)[b] Yield (%)[b] 1 Et3N 86 18 2 DIPEA 92 32 3 Cy2NMe 95 26 4 NaCO3 98 58 5 K2CO3 95 21 6 Cs2CO3 93 10 7 K3PO4 100 70 [a] [b] All reactions were conducted on a 0.2 mmol scale, using 1.0 equiv. phenylboronic acid. Determined by GC with docosane as internal standard.
The ratio of reagents and additives were also examined, and the investigation showed that the catalytic cycle was unreactive at 1 mol% loading (Table 8-4, entry 1), and incomplete at 2 mol% (Table 8-4, entry 2). At 3 mol%, however, the yield was quantitative (Table 8-4, entry 3), which suggests that 5 mol% loading of the catalyst was wasteful. Surprisingly, reducing the equivalents of base increased the yield to a quantitative transformation with 5 mol% of the catalyst (Table 8-4, entry 4). Following suit, when the amount of base was reduced down to 0.5 equivalents (66 %, Table 8-4, entry 6), this produced a better yield than that at 1.0 equivalents (25 %, Table 8-4, entry
5). The quantitative yield was retained when the K3PO4 was used at 2.0 equivalents with 3 mol% of the catalyst and ligand; there were no deleterious effects when the ratio between the catalyst and ligand was changed either. The optimised conditions (Table 8- 4, entry 7) corresponded to an isolated yield of 76 %.
138 | Chapter 8: Suzuki – Miyaura Cross - Coupling Reactions
Table 8-4. Role of the ratio of reagents in the Suzuki cross-coupling reaction between 4-cyanotosylate (251) and phenylboronic acid (250).
(dppf)Ni[P(O-p-tol) ] DPPB K PO Conversion Yield Entry 3 2 3 4 (mol%) (mol%) (equiv.) (%)[b] (%)[b] 1 1 1 4.0 0 0 2 2 2 4.0 92 28 3 3 3 4.0 100 100 4 5 5 2.0 99 100 5 5 5 1.0 70 25 6 5 5 0.5 71 66 7 3 3 2.0 100 100 8 3 5 2.0 100 100 [a] [b] All reactions were conducted on a 0.2 mmol scale, using 1.0 equiv. phenylboronic acid. Determined by GC with docosane as internal standard.
8.5 Scope of aryl tosylate coupling partner with phenylboronic acid. With the optimised conditions in hand, the substrate scope was explored next. All aryl tosylates in this section were synthesised by treating the corresponding phenols with tosyl chloride according to literature procedures (c.f. Chapter 10).433 The neutral phenyl tosylate was well tolerated and produces the biphenyl product (252) in 69 % yield. Most of the other functionalised tosylates did not perform as well, regardless of the electron donating or withdrawing nature of their substituents. No reaction occurred for any substituents containing a nitrogen group directly bonded to the phenyl ring (amino group, 253 and nitro group, 254); the result that both strongly electron withdrawing and activating coupling partners were not tolerated in this system is noted. The slightly less activating methoxy substituted (255) and less deactivating trifluoromethyl substituted (256) phenyltosylates produced yields of 40 % and 43 %, respectively. The weakly activating, electron-donating phenyl group gave a 43 % yield of the triphenyl (257), whereas the 4-tert-butyl produced a yield of 59 % (258). It is interesting to note that the slightly more electron donating 3,5-di-tert-butyl derivative was entirely unreactive (259). Many of these derived tosylates have been shown to couple with other nickel catalysts prior417,434 and following this investigation.435
8.5 Scope of aryl tosylate coupling partner with phenylboronic acid. | 139
Table 8-5. Scope of the aryl tosylate coupling partner 267.
[a] All reactions were conducted on a 0.5 mmol scale, using 1.0 equiv. phenyltosylate coupling partner.
A small investigation into the steric effects of the phenyltosylates showed that when a methyl group is substituted in the para- position, the yield achieved was 53 % (260), however, when the methyl group is substituted in the ortho- position, the yield was reduced to 31 % (261). When there were methyl groups in the para- and both ortho- positions, there was no reaction (262); therefore, it was deduced that steric hindrance has a negative effect on the coupling between phenyl tosylate and phenyl boronic acid as described in previous investigations in Suzuki cross-couplings.417 Following this publication, Buchwald reported a catalyst for sterically encumbered aryl halides.436 It was noted that the 2,6-dimethoxy phenyl tosylate underwent the coupling successfully as previously discussed, however, this could be due to the strongly activating electron- donating nature of the substituents.
140 | Chapter 8: Suzuki – Miyaura Cross - Coupling Reactions
Competition between halogens and tosylates were examined next, using 3- bromophenyltosylate and 4-iodophenyltosylate. In both cases, the triphenyl products were isolated (263 and 264), as well as the mono-coupled products (265 and 266); these couplings showed that the halogens were more reactive than the tosylate group. This result was not surprising, as studies have shown that the rate of displacement of tosyl groups is slower than that of bromide and iodide ions.437 This is due to the comparatively lower bond-dissociation energies of C-X than that of C-O,.438 as well as the oxophillic nature of nickel, which can form stronger bonds with oxygen than with halogens (apart from Ni-F).439 From 3-bromophenyltosylate, the triphenyl (263) was produced at 45 % yield, compared to 30 % of the 3-tosyl diphenyl (265). Surprisingly, yields from the 4-iodophenyltosylate were lower at 28 % (triphenyl, 264) and 37 % (4- tosyl diphenyl, 266).
8.6 Scope of the aryl boronic acid coupling partner with 4- cyanophenyltosylate. Next, the scope of the phenylboronic acid coupling partner was examined using 4- cyanophenyltosylate. Similar to the tosylate scope, as well as the substrate scope of the Mizoroki–Heck reaction in 7.4.2, the presence of a nitrogen functionality within the boronic acid/ester coupling partner led to no reaction (268, 269, 270). At this point, it is important to note that scope limitations of substrates bearing exposed Lewis basic nitrogens has been previously reported,440 with suggestion that these substrates render the reaction fruitless due to catalyst poisoning.441 However, successful nickel-catalysed cross-coupling of nitrogen-rich heterocycles has also been reported.442
Table 8-6. Scope of the aryl boronic acid coupling partner 280.
8.6 Scope of the aryl boronic acid coupling partner with 4 - c y a n o p h e nyltosylate. | 141
[a] All reactions were conducted on a 0.5 mmol scale, using 1.0 equiv. phenylboronic acid coupling partner. *from boronic acid pinacol ester
The slightly electron-withdrawing 4-carboxyphenyl boronic acid produced no reaction (271); this is surprising, since the relatively moderately deactivating partners were well tolerated in the aryl tosylate scope. Two plausible explanations may be caused the interference of its carboxylic acid moiety: 1) the system may be rendered unreactive by its likely interaction with K3PO4, thereby using up amounts of base that is important in the proceedings of the catalytic cycle; or 2) its deprotonation by K3PO4 into the carboxylate ion, which may not be well tolerated in such a system. To the best of our knowledge, 4-carboxyphenyl boronic acid has never been shown to be cross-coupled with para- substituted arylboronic acids.
Investigations into the electronic effects of substituents on phenylboronic acid showed that both electron-donating and –withdrawing substrates were well tolerated, apart from the strongly activating nitrogen-containing molecules. The electron-rich 4- biphenylboronic acid and 4-methoxyboronic acid performed well to produce their corresponding products at 72 % (272) and 71 % (273), respectively. Good yields were acquired from the electron-withdrawing boronic acid partners 4- 142 | Chapter 8: Suzuki – Miyaura Cross - Coupling Reactions methyoxycarbonylphenylboronic acid (274, 67 %), 4-(trifluoromethyl)phenylboronic acid (275, 75 %) and 4-fluorophenylboronic acid (276, 72 %).
Steric effects were also examined next by comparing the activity of ortho-, meta-, and para-methyl phenylboronic acids. The ortho-, and para- products (277 and 278) produced comparable yields of 79 % and 76 %, respectively, to that of biphenyl (249, 76 %). The meta-methyl product (279) offered a slightly reduced yield of 64 %. The difference in yield may be explained by the fact that alkyl substituents are known to be ortho/para directing functional groups.443 It is noted that in Monteiro’s study, the opposite effect was observed in the same reactions, where the meta-methyl product was isolated at a higher yield of 88 % compared to yields of 60 % and 79 % of the ortho-, and para-methyl products.417
In this investigation, we have shown that it is possible to catalyse Suzuki–Miyaura reactions with our series of nickel(0)-phosphites and phosphite-phosphate complexes. A fair range of functional groups were well tolerated in this reaction with the optimised
(dppf)Ni[P(O-p-tol)3]2/DPPB system. We were able to increase the yield for the reaction between phenyl boronic acid (250) and 4-cyanophenyltosylate (251) from the preliminary value of 31 % up to 76 %. While this improvement was not comparable to Monteiro’s high yield of 96 %,417 our investigation presents series of five new air-stable nickel(0) phosphite-phosphate complexes that are capable of catalysing the reaction between aryl tosylates and aryl boronic acid into the current literature.
N.B. Concurrent to the time of our work, a report published by Doyle et al.385 described an analogous study on a modular, air-stable nickel precatalyst 281. This complex was also proposed to allow the addition of ligands to form a more reactive Ni-phosphine system. In this work, the Doyle group was able to produce near quantitative 98 % yield for the reaction between 250 and 251.
Figure 8-2. Structure of moldular, air-stable nickel precatalyst by Doyle et al.385 8.6 Scope of the aryl boronic acid coupling partner w i t h 4 - cyanophenyltosylate. | 143
Chapter 9: Summary and conclusions- Part II The second part of this thesis described the use of new air stable nickel(0) catalysts in cross-coupling reactions. The purpose of this investigation was to explore the catalytic activity of homoleptic nickel(0)phosphite complexes Ni[P(OPh)3]4 and Ni[P(OEt)3]4 and a series of five new air stable phosphine-phosphite nickel(0) complexes (Figure 7-1, Scheme 7-3) as possible alternatives to widely-used but costly palladium catalysts and air sensitive nickel(0) sources Ni(cod)2 and (PCy3)2NiCl2.
Initially, the study investigated the suitability of these Ni(0) complexes for the in the Mizoroki–Heck cross-coupling of aryl triflates (239) with an electron-rich olefin (206).
Of these compounds, (dppf)Ni[P(OPh)3]2 was the most efficient catalyst for these C-C coupling reactions. Unfortunately, the substrate scope was very limited, with only four examples out of thirteen that successfully formed new C-C bonds (Table 7-5). Reactivity of phenylhalides and pseudohalides were also trialled with little success. This nickel-based catalytic system was also trialled as a potential replacement of the palladium system used in the main cross-coupling reaction for the antibacterial compound 135C. However, this reaction was deemed unsuccessful.
Scheme 9-1. A new air-stable nickel(0) phosphite complex for the Mizoroki–Heck cross-coupling reaction between aryl triflates and vinylbutyl ether.
Next, the same catalysts were tested in Suzuki–Miyaura cross-coupling reactions between arylboronic acids and aryl pseudohalides. Of these compounds, (dppf)Ni[P(O- p-Tol)3]2 was the most efficient catalyst for these C-C cross-coupling reactions. Amongst 13 different aryl tosylates (Table 8-5), most weakly activating electron- donating and neutral substrates performed well with moderate to good yields between 43 – 69 %. Substrates with electron-withdrawing groups, and substrates with steric hindrance afforded the products in lower yields (28 – 45 %). Any substrate that contained nitrogen in its structure presented with no reaction. Competition between 144 | Chapter 9: Summary and conclusions - P a r t I I halogens and tosylates showed that the halogens were more reactive than the tosylate group.
Scheme 9-2. A new air-stable nickel(0) phosphite complex for the Suzuki–Miyaura cross-coupling reaction between arylboronic acids and aryl tosylates. Scope summary of the aryl tosylate coupling partner 267.
Similar to the tosylate scope, the presence of nitrogen atoms in the structure of the boronic acid/ester coupling partner led to no reaction. The investigation showed no significant difference in the yields between the neutral, electron-donating and electron- withdrawing substituted boronic acids (Table 8-6).
Scheme 9-3. A new air-stable nickel(0) phosphite complex for the Suzuki–Miyaura cross-coupling reaction between arylboronic acids and aryl tosylates. Scope summary of the boronic acid coupling partner 280.
In conclusion, it was shown that air-stable nickel(0) phosphite catalysts can be used as viable alternatives to the commonly used air-sensitive Ni(0) sources and expensive palladium catalysts. In the current phase of the investigation, there are limitations with respect to narrow substrate scopes, particularly in the Heck cross-coupling reaction. However, these phosphine-phosphite complexes are viable at a low catalytic loading (3 mol%), and are more economical than other air-stable alternatives. The nature of their trivial preparation may also allow for simple modifications of both the phosphine and the phosphite ligands in the future, perhaps leading to new complexes that may be able to address the current limitations. 8.6 Scope of the aryl boronic acid coupling partner w i t h 4 - cyanophenyltosylate. | 145
146 | Chapter 10: Experimentals
Chapter 10: Experimentals 10.1 Chemistry general protocol Starting materials and reagents were purchased from Sigma-Aldrich or Merck chemical companies. N-Methyldicyclohexylamine was distilled under reduced pressure and stored under argon. Pd2(dba)3∙CHCl3, and Pd(PPh3)4 were prepared as previously described. All reactions were performed under argon and at ambient temperature unless stated otherwise. All solvents used in reactions were anhydrous unless noted otherwise. Anhydrous solvents were distilled over the appropriate drying agent or acquired from a Pure Solv 5-Mid Solvent Purification System (Innovative Technology Inc.).
10.2 Instruments and materials 10.2.1 Chemistry 1H and 13C Nuclear Magnetic Resonance (NMR) spectra were acquired on a Varian 300, Varian 400, Bruker AV500 or a Bruker AV600 spectrometer and all signals δ are reported in parts per million (ppm). 1H and 13C assignments where indicated were made with the aid of DEPT, COSY, HSQC and HMBC sequences. Chemical shifts were referenced to the residual (partially) undeuterated solvents and reported in parts per million (ppm). Infrared spectra were collected neat on a PerkinElmer Spectrum One FT- IR spectrometer as neat samples and recorded in wavenumbers (cm-1) at 2 cm-1 resolution.
Mass Spectra were acquired on VG Autospec spectrometer using electrospray ionization (ESI) or atmospheric pressure ionization (API). HRMS was performed with a standard resolution of approximately 10,000. Melting points were recorded on a Reichart heated- stage microscope.
All synthetic reactions were performed under argon unless otherwise noted. Thin Layer Chromatography (TLC) was performed on Analtech silica gel 60 F254 pre-coated aluminium sheets. Visualisation of TLC plates was achieved with a 254 or 365 nm UV lamp. Column chromatography was performed using SiliaFlash® 60 (40 – 63 μm) supplied by SiliCycle. Chromatography solvents were distilled prior to use. Anhydrous solvents were distilled over the appropriate drying agents444 or by the solvent purification system (Pure Solv., Innovative Technology, Inc.). Starting materials and reagents were generally available from Sigma Aldrich or Alfa Aesar. 10.2 Instruments and materials | 147
10.2.2 Microbiology Bacterial strains: Bacteria and fungi were obtained from the culture collection of The School of Biomedical Sciences at The University of Western Australia (UWA). Staphylococcus aureus strains: NCTC 10442, NCTC 6571, 1045638Y, 1045127J, 3754546E, 380733M, 3905934A, 4256936D, RPH 15256, 1045455M, RPH 15358, RPH 15879, RPH 15515, RPH 15913, RPH 15067, RPH 15185, RPH 15046, RPH 15056, 1045430U, 21742M; Staphylococcus epidermidis strains: P1, Q2, T1, 21137, 26025, 21248, 26124, 27314, 21249, 21081; Staphylococcus hominis P2; S. hominis 5219; Staphylococcus warneri 4863; S. warneri A1; Staphylococcus saprophyticus 4753; S. saprophyticus 8213; Staphylococcus capitis F2; S. capitis 4818; Staphylococcus haemolyticus 4865; S. haemolyticus Q1; Micrococcus sp. NCTC 2665, U2, I3, M384, M2916,Q35, 2063,M371, M2064, 4349; Streptococcus pyogenes strains: ATCC 10389, 4557, 4026, 4639, 8402, 7963, 4197, 8469, 6417, 7662; Streptococcus pneumoniae strains: 2409, 2403, 2406, 2403, 2411, 2401, 2431, 2415, 2418, 2419; Moraxella catarrhalis strains: M3584, M3586, M3587, M3588, M3589, M3609, M3610, M3611, M3612, M3613; Clostridium difficile strains: ANSW 85, ANSW 87, NCTC 46593, ANSW 70, WA 189, ANSW 137, ANSW 192, ANSW 72, ANSW 133, WA 187, WA 191, WA 194.
Consumables: Disposable plasticware, such as pipette tips and transfer pipettes, were manufactured by Samco Scientific Co., and Eppendorf. Petri dishes (90 mm) and 96- well microtitre trays were manufactured by Becton Dickinson Company; sterile 0.22 μM pore filters were manufactured by Pall Corporation.
Instrumentation: Optical density measurements were collected on a Bio-Rad xMark Microplate Spectrophotometer at 600 nm. Turbidity adjustments were made using a colorimeter (Hatch Company, Loveland, Colorado USA).
Media Preparation: Blood agar was supplied by PATHWEST Media, Mount Claremont, Western Australia. Mueller Hinton Agar and Mueller Hinton Broth were purchased as powders and prepared according to the manufacturers’ instructions.
Compounds for testing: Compound 135C was synthesised as described previously94 and purity was confirmed by 1H and 13C NMR spectroscopy. The yellow solid was stored 148 | Chapter 10: Experimentals under argon and protected from light at -20 °C. Stock solutions of 135C in DMSO were prepared fresh on each testing occasion and diluted as required. The maximum concentration of DMSO present in bioactivity assays was 10%. Antimicrobial agents: Stock solutions of all novel compounds were made by dissolving each in DMSO at 10 mg/mL.
10.3 Experimental procedures- Part I 10.3.1 Biological assays 10.3.1.1 Susceptibility tests Disc diffusion method: The disc diffusion method was based on that recommended by the Clinical and Laboratory Standards Institute (CLSI, 2012a).445 Briefly, inocula were prepared by suspending growth from fresh cultures on blood agar in 0.85 % saline and adjusting to a cell concentration of approximately 108 colony forming units (cfu) per ml (0.5 McFarland turbidity). Cell suspensions were then swab-inoculated onto Mueller- Hinton (MH) agar plates (Oxoid Ltd., Hampshire, England). A blank 6 mm AA paper disc (Whatman Ltd., England) was placed in the centre of each plate and 20 µL of the antimicrobial agent (10 mg/mL) was pipetted onto the disc. Cultures were then incubated at 37oC for 24 h under the appropriate conditions. After incubation, zones of inhibition were measured to the nearest millimetre. Assays were performed three times per organism/derivative combination.
Broth micro-dilution method: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined using methods recommended by the Clinical and Laboratory Standards Institute (CLSI 2012b).446 Briefly, each compound was serially diluted two-fold in 100 µL volumes in a 96-well microtitre tray (Falcon, Becton Dickinson, USA). Inocula were prepared by adjusting cell suspensions prepared from overnight blood agar cultures to 0.5 McFarland turbidity standard and then diluting 1 in 100 to give a cell suspension of approximately 106 cfu/mL. Each microtiter well was inoculated with 100 µL of a bacterial inoculum and the trays were incubated at 37 °C for 24 h or 48 h for anaerobes, under the appropriate conditions.
For a subset of Gram-negative bacteria, MICs were also determined in the presence of the cell membrane permeabiliser polymyxin B nonapeptide (PMBN), at a final concentration of 5 μg ml-1.MICs were determined as described above with the following adjustment: 135C was serially diluted two-fold in 50 μL volumes in a 96-well microtitre 1 0 . 3 E x p e r imental procedures - P a r t I | 149 tray (Falcon, Becton Dickinson, USA), after which 50 μL of PMBN at 20 μg ml-1 was added to each microtitre well. Novobiocin(0.5 – 512 μg ml-1) was used as a positive control188 for assays with PMBN.
MICs were determined visually as the lowest concentration of antibacterial preventing growth. MBCs were determined by subculturing 10 µL volumes from non-turbid wells, spot-inoculating onto blood agar and incubating for 24-48 h. The lowest concentration of antibacterial with no resultant growth was determined as the MBC. MBCs were not determined for anaerobic bacteria nor the assay with PMBN. This assay was performed in triplicate per organism/derivative combination. Where results differed the higher value was selected as the final result.
10.3.1.2 Haemolytic activity assay As an indicator of cytotoxicity, the haemolysis of sheep erythrocytes by compounds 10 and 17 was investigated. Master stock solutions of compound 10 (10 mg/ml) were made by dissolving 20 mg of the dehydrated compound in 2 ml 100 % DMSO. They were stored in foil-covered glass bottles at -20 °C. Solutions stored in this way retained full antimicrobial activity for a minimum period of 6 weeks (results not shown). Serial 10- fold dilutions of compounds 10 and 17 were performed in phosphate buffered saline (PBS) to make solutions of 1000, 100, 10 and 1 μg/mL. In microcentrifuge tubes, 500 µL of each dilution was combined with 480 µL PBS and 20 µL washed sheep erythrocytes (100%) so that the final concentration of erythrocytes was 2 % and the final concentrations of compound were 500, 50, 5 and 0.5 μg/mL. Dilutions of DMSO alone were prepared and tested as above to check for haemolysis due to DMSO. A positive control (100% haemolysis) was prepared with 980 µL water and 20 µL erythrocytes. A negative control was prepared with 980 µL PBS and 20 µL erythrocyte suspension. Tubes were incubated at 37 °C for 2 h on a rocker then centrifuged at 12 000 g for 5 mins. The optical density of the supernatant was determined by transferring 100 µL volumes of each reaction to a microtitre tray then reading the OD at 540 nm. Percentage haemolysis was determined by blanking the OD against that of the negative control and presenting the resulting OD as a proportion of the OD of the positive control (blanked with water). All dilutions and controls were prepared in duplicate and the entire assay was performed three times on separate occasions.
150 | Chapter 10: Experimentals
10.3.1.3 Cytotoxicity assay The cytotoxocity of compound 10 was assessed using an in vitro cytotoxicity assay. Mammalian fibroblast L929 cells grown to approximately 80% confluency in HGM-M, were washed with Hanks, trypsonised to detach the cells from the flask, then diluted to 105 cells/mL in HGM-M. Volumes of 200 µL were used to inoculate the wells of a 96- well microtitre tray. After incubation for 24 h at 37 °C, adherent cells were washed with Hanks and then 100 µL of HGM-M was added to the wells. Compound 10 (10 mg/mL in DMSO) was serially 10-fold diluted in HGM-M to make solutions of 1000, 100, 10 and 1 µg/mL. Volumes of 100 µL of each solution were then added to the wells of the microtitre tray giving final concentrations of 500, 50, 5 and 0.5 µg/mL. Equivalent dilutions of DMSO were prepared and tested to check that cytotoxic activity was not due to the DMSO. A negative control was prepared containing only HGM-M, and a positive control was prepared by adding 100 µL of carboplatin (10 mg/mL; Mayne Pharma Pty Ltd, Australia) to wells containing 100 µL HGM-M. Controls and dilutions of compound 10 and DMSO were prepared in duplicate. After 24 h incubation at 37 °C with shaking, cytotoxicity was quantified using the neutral red assay. The cells were washed with Hanks then 200 µL HGM-M and 20 µL Neutral Red (3.3 g/L; Sigma- Aldrich) was added to each well. After 2 h incubation at 37 °C with shaking, cells were washed twice with PBS then 200 µL of 1 % acetic acid in 50 % EtOH was added to each well to solubilise the stain. After 15 min incubation at 37 °C with shaking the optical densities of the wells at 690 nm was determined and subtracted from the OD540. Values were then blanked against wells to which no cells had been added, and converted to a ratio of the OD of the negative control. Ratios of ≤0.5 indicated a cytostatic effect. Testing was performed on two separate occasions.
10.3.1.4 Ames test The Ames test for mutagenicity was based on the method published by Zeiger and Mortelmans,193 in the absence of a metabolic activation system. To prepare inocula, single colonies from overnight BA cultures of three commonly used Salmonella tester strains, S. typhimurium TA98, TA100 and TA1535, were used to inoculate 10 mL of nutrient broth. After incubation for 15-18 h at 37 °C with shaking, the concentration of the culture was appropriate for use in the test (~1-2 × 109 cfu/mL). Glucose minimal agar plates (20 mL volume) were dried thoroughly. Molten top agars (2 mL) were prepared, supplemented with biotin and trace histidine, and maintained at 43-48 °C before adding 50 µL of the bacterial culture (~1×108 cells) and 100 µL of test solution 10.3 Experimental procedures - P a r t I | 151
(see below). The molten top agar was then poured directly over the surface of the glucose minimal agar and gently swirled to ensure even distribution. Once solidified, plates were incubated at 37 °C for 48 h then bacterial colonies were counted. Test solutions were prepared as follows and included three concentrations of compound 10 or 17, a negative solvent control and a positive control (selected from the recommended positive control chemicals and test concentrations) for each strain. Compounds 10 and 17 were assessed by incorporating 100 µL of the stock solution (10 mg/mL in DMSO) directly into molten top agar to test the compound at 1000 µg/plate. Dilutions of the stock were prepared in sterile distilled water to also test 300 µg/plate and 100 µg/plate. DMSO (100 µL of 100 %) was incorporated into molten top agar as the negative solvent control. Positive controls were 4-nitro-o-phenlenediamine at 2.5 µg/plate (for TA98), and sodium azide at 5 µg/plate (for TA100 and TA1535). A mutagenic effect is indicated when colony counts are two to three times greater with test compound than on the negative solvent control plate and this is regarded as ‘positive’. In these cases, the increase in colonies is usually dose related. A positive result in this test is highly predictive of rodent carcinogenicity. Testing was performed in duplicate on two separate occasions.
10.3.1.5 Determination of antimicrobial synergy Minimum inhibitory concentrations of 135C in combination with ciprofloxacin, rifampicin, vancomycin, erythromycin, gentamicin and oxacillin against S. aureus NCTC 6571were determined to investigate whether synergy or antagonism occurred. Each antibiotic was serially diluted two-fold in 50 μL volumes in a 96-well microtitre tray (Falcon, Becton Dickinson, USA), after which 50 μL of 135C at varying concentrations (ranging from 0.06 -4 μg ml-1) was added to each dilution series. The remainder of the assay was performed as described above. Fractional inhibitory concentration index (FICI) values were determined for each combination using the equation: FICI = FICA + FICB = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone).This assay was performed three times per antibiotic/135C combination.
10.3.1.6 Cellular leakage and lysis Inocula were prepared from an overnight culture of S. aureus NCTC 6571 in MHB by diluting to 0.5 McFarland in fresh MHB. Bacterial suspensions were then left untreated 152 | Chapter 10: Experimentals
(untreated control) or treated with 2, 32 (1×MBC) and 320 μg ml-1 (10×MBC) 135C. Treatments and the control were incubated at35°C with shaking. Samples were removed immediately after inoculation (time 0), and further samples were taken at 15, 30, 60, 120 and 240 min. Cell lysis was determined by measuring the optical density (OD) of each sample at 600 nm. The leakage of intracellular contents was determined by filtering samples (0.45 μm) to obtain cell-free filtrates and determining the OD260at time zero and 240 min only. The OD of all samples was determined against the appropriate blank at the appropriate wavelength. Each assay was performed three times and mean values were determined.
10.3.1.7 Time-kill kinetics Inocula were prepared as described for the cell leakage and lysis assays and exposed to 135C at 0 (untreated control), 32 and 320 μg ml-1 of 135C in MHB. Treatments and controls were incubated at 35°C with shaking and samples were removed at 0, 15, 30, 60, 120 and 240 min. Samples were immediately serially diluted ten-fold in 0.01 M phosphate buffered saline at pH 7.0 (PBS) and 10 μL aliquots were spot-inoculated in duplicate onto MHA. Agar plates were incubated at 37 °C overnight before determining the viable count. The assay was conducted on two separate occasions.
10.3.1.8 Serial passage with compound 135C to evaluate resistance development Four S. aureus strains were investigated for the development of multi-step resistance to 135C by serially passaging in increasing concentrations of the compound. Briefly, MICs were determined using the CLSI broth microdilution method as described above.
Serial passage was performed by removing the contents from wells with OD600values of approximately 0.1, and using to inoculate a fresh dilution series. This was repeated at 24 h intervals for a total of 10 passages. The compound concentration range of each new passage was based on the MIC from the previous passage. At the end of the serial passage experiment, isolates were passaged daily on BA and MICs for 135Cwere determined again after 3 and 10 drug-free passages. After 3 drug-free passages, MICs were also determined for ciprofloxacin, erythromycin, gentamicin, oxacillin, rifampin, vancomycin, kanamycin and chloramphenicol to evaluate whether cross-resistance was evident. The entire experiment was performed in duplicate on two separate occasions. 10.3.1.9 Relative fitness of S. aureus isolates serially passaged with 135C To evaluate relative fitness, S. aureus serial-passaged isolates and parent strains were cultured overnight on brain heart infusion agar (BHIA) plates, then 2-3 well-isolated 10.3 Experimental procedures - P a r t I | 153 colonies were inoculated into 15 mL of brain heart infusion broth (BHIB) and incubated at 37°C with shaking at 80 rpm. After overnight growth, cultures were diluted to an
OD600 of 0.01 and 150 μL of this was added to 15 mL of BHIB to give an inoculum concentration of approximately106 CFU ml-1. Cultures were incubated at 37 °C with shaking at 125 rpm for 24 h. The OD600of cultures was determined every hour for 1-7 h and then at 24 h. The entire assay was performed in duplicate twice.
10.3.1.10 Genomic DNA preparation and whole genome sequencing Following subculture of S. aureus isolates on blood agar for 24 h, genomic DNA was extracted using a Gentra Puregene Yeast/Bact. Kit [Qiagen, Hilden, Germany] and multiplexed paired-end (PE) libraries were generated using standard Nextera XT protocols [Illumina, CA, USA]. Whole genome sequencing (WGS) of eight S. aureus strains (4 generated mutants, 4 wildtype) was performed using a MiSeq benchtop sequencer [Illumina], generating 300 bp PE reads. Sequence data for this study has been deposited in the European Nucleotide Archive (ENA) under study PRJEB21492 (sample accessions ERS1797685-ERS1797692, see Supplementary Table 1). To examine genetic differences between wildtype parent strains and mutant progenitors, high-resolution single nucleotide variant (SNV) analysis was performed, as previously described 206. The highly annotated genome of S. aureus strain MRSA-252 [Genbank accession BX571856, multilocus sequence type (ST) 36] was used as a reference chromosome for read mapping (to a median depth of 45X across the 2.9 Mbp chromosome, range 26.9-52.6X) and SNV calling.447 . 10.3.2 Protein Crystallography 10.3.2.1 Procedure for the removal of fatty acids from serum albumin Bovine serum albumin (1.0 g) was dissolved in 10 mL of distilled water at RT. Activated charcoal (0.5 g) was stirred into the solution, and the pH was adjusted to 3.0 by the addition of 0.2 M HCl. The solution was stirred for 1 h at 0 °C, followed the removal of the charcoal by centrifugation at 20,000 x g for 30 min in a Heraeus Multifuge X3R with an [rotor] rotor at 4 °C. The clarified solution was then neutralised to pH 7.0 with 0.2 M NaOH. The solution was centrifuged at 24,000 x g for 15 min at 4 °C to remove formed salts; it was then filtered (0.2 µm filter) to remove any remaining particles.162
10.3.2.2 Procedure for the purification of serum albumin 154 | Chapter 10: Experimentals
After the removal of fatty acids from serum albumin, the solution was concentrated to approx. 50 mg/mL. Gel-filtration chromatography on an AKTA FPLC system (brand plz) was carried out using a XX method in buffer (100 mM NaCl and 10 mM Tris at pH 7.4). An SDS-PAGE was performed to confirm the purity of the protein.
10.3.2.3 Preparation and crystallisation of the complexes of bovine serum albumin The complex of BSA with 135C was formed by mixing defatted and purified albumin at 1 mM (67 mg/mL) concentration in buffer (100 mM NaCl and 10 mM Tris at pH 7.4) with 10-molar excess of 135C, which was added in the form of 200 mM solution in ethanol. The mixture was incubated in 28 °C with shaking and was centrifuged before crystallisation setup in two conditions: (1) 18% PEG MME 5K, 0.2M CaOAc, 0.1M MES pH 6.9; and (2) 20% PEG MME 5K, 0.2M CaOAc, 0.1M MES pH 6.7.160 The vapour diffusion hanging drop method was used for crystallisation of the protein.
10.3.3 Saturation Transfer Difference NMR All NMR spectra were recorded on a Bruker AV3HD 600 MHz spectrometer equipped with a 5 mm z-gradient BBFO probe at 298 K. All spectra were processed with Topspin 3.5. The duration of the 1H 90° pulse was 9.60 µs. The samples for STD experiments contained 1 mM 135C and 20 µM bovine serum albumin (positive control only) in 600
μL 7% DMSO-d6/phosphate buffer at pH 6.8. An additional 60 μL of D2O was added to the samples prior to the experiment. The 1H STD spectrum was obtained using 4096 scans (2048 scans each for on- and off-saturation) using the Bruker STDDIFFGP19.3 pulse sequence. On-resonance and off-resonance saturation at -3.0 and -30 ppm respectively used a 20 ms Gaussian excitation pulse comb applied for 2 s with an excitation band as described elsewhere.448
10.3 Experimental procedures - P a r t I | 155
10.3.4 Synthesis 10.3.4.1 2-(4-Vinylphenyl)acetonitrile (59)
To an ice-cooled solution of 18-crown-6-ether (0.14 g, 0.52 mmol) in anhydrous acetonitrile (12 mL), 4-vinylbenzyl chloride (1.84 mL, 13.1 mmol) and potassium cyanide (1.28 g, 19.6 mmol) was slowly added. The solution was stirred at room temperature for 20 h before being concentrated in vacuo and diluted with water (20 mL).The solution was then extracted with diethyl ether (3 x 15 mL) and the organic layer was washed with water (10 mL) and brine (10 mL), dried with Na2SO4 and filtered. The solvent was removed under reduced vacuum and the crude mixture was purified by column chromatography (1:9 ethyl acetate/hexane) to afford a yellow oil (1.54 g, 82 %). The spectral data for this compound matched those reported in literature.114 1 H NMR (400 MHz, CDCl3): δ 7.27 (d, J = 8 Hz, 2H, H4/H6), 7.14 (d, J = 8 Hz, 2H,
H3/H7), 6.56 (dd, J1 = 8 Hz, J2 = 16 Hz, 1H, H8), 5.62 (d, J = 16 Hz, 1H, H10), 5.14 (d, J
= 8 Hz, 1H, H9), 3.59 (s, 2H, H1).
10.3.4.2 2-(4-Vinylphenyl)acetic acid (56)
To a solution of 2-(4-vinylphenyl)acetonitrile (59) (1.53 g, 10.7 mmol) and hydroquinone (23.5 mg, 0.21 mmol) in 95 % ethanol (15 mL), potassium hydroxide (4.19 g, 42.8 mmol) is added. The solution is refluxed for 12 h. After cooling, water (10 mL) was added and the solution was extracted with ether (2 x 15 mL). The aqueous 156 | Chapter 10: Experimentals solution was acidified with conc. HCl until pH ≈ 1 and was then extracted with chloroform (3 x 10 mL), dried with MgSO4 and filtered. The solvent was removed under reduced pressure to afford a pale yellow oil (1.55 g, 90 %). The spectral data for this compound matched those reported in literature.114 1 H NMR (400 MHz, CDCl3): 7.38 (d, J = 7.8 Hz, 2H, H4/H6), 7.25 (d, J = 10.4 Hz, 2H,
H3/H7), 6.70 (dd, J1 = 17.6 Hz, J2 = 10.4 Hz, 1H, H8), 5.73 (d, J = 17.6 Hz, 1H, H9),
5.24 (d, J = 10.8 Hz, 1H, H10), 3.64 (s, 2H, H1).
10.3.4.3 Methyl 2-(4-vinylphenyl)acetate (54)
To a solution of 2-(4-vinylphenyl)acetic acid (56) (0.77 g, 4.757 mmol) in THF (5 mL), DBU (0.92 mL, 6.18 mmol) was slowly added at 0 °C and stirred for 10 min. Methyl iodide (0.38 mL, 6.18 mmol) was then added dropwise to the solution which was then stirred at room temperature for 3 h. The solution was diluted with diethyl ether (5mL) and washed with 5mL of each of the following: H2O, 2M HCl, 2M NaOH, 2M HCl and
H2O. The organic layer was dried (Na2SO4), filtered and dried under vacuum to afford a clear oil (0.68 g, 81 %). The spectral data for this compound matched those reported in literature.107 1 H NMR (400 MHz, CDCl3): 7.37 (d, J = 7.3 Hz, 2H, H4/H6), 7.24 (d, J = 7.3 Hz, 2H,
H3/H7), 6.70 (dd, J1 = 17.6 Hz, J2 = 12 Hz, 1H, H8), 5.73(d, J = 17.6 Hz, 1H, H9), 5.23
(d, J = 12 Hz, 1H, H10), 3.69 (s, 3H, H11), 3.62 (s, 2H, H1).
10.3 Experimental procedures - P a r t I | 157
10.3.4.4 Trimethyl 2,2',2''-(((1E,1'E,1''E)-benzene-1,3,5-triyltris(ethene-2,1- diyl))tris(benzene- 4,1-diyl))triacetate (67)
Tribromobezene (250 mg, 0.79 mmol), Pd2(dba)3∙CHCl3 (41.1 mg, 0.039 mmol) and [(t-
Bu)3PH]BF4 (11.52 mg, 0.039 mmol) were dissolved in dry DMF (2 mL). Cy2NMe (0.67 mL, 3.16 mmol) and methyl 2-(4-vinylphenyl)acetate (54) (461.8 mg, 2.62 mmol) were added and the reaction mixture was subjected a freeze-pump-thaw cycle three times. The resultant solution was stirred at 80°C for 36 h. After cooling, water (5 mL) was added and the solution was extracted with CH2Cl2 (3 x 5 mL). The combined organic extracts were washed with water (3 x 5 mL) and brine (5 mL), dried with
MgSO4 and filtered. The organic layer was concentrated under reduced pressure to give a yellow residue. The residue was subjected to column chromatography (1:19 ethyl acetate/toluene) to afford a bright yellow oil (0.45 g, 82 %)
Rf = 0.17 (20 % ethyl acetate/hexane) 1 H NMR (400 MHz, CDCl3): 7.55 (s, 3H, H2), 7.52 (d, J = 8.2 Hz, 6H, H5’/H7’), 7.30 (d,
J = 8.2 Hz, 6H, H4’/H8’), 7.19 (d, J = 16.3 Hz, 3H, H2’), 7.13 (d, J = 16.3 Hz, 3H, H1’),
3.72 (s, 9H, H11’), 3.66 (s, 6H, H9’). 13 C NMR (100 MHz, CDCl3): 172.1 (C10’), 138.2 (C3’), 136.3 (C1), 133.6 (C6’), 129.8
(C5’/C7’), 129.0 (C2’), 128.5 (C1’), 126.9 (C4’/C8’), 124.1 (C2), 52.3 (C11’), 41.1 (C9’). IR (neat): 3026, 2950, 1730 (C=O), 1585, 1512, 1434, 1153. + HR-MS (ESI): C39H37O6 [M + H] calcd 601.2590, found 601.2590.
158 | Chapter 10: Experimentals
10.3.4.5 2,2',2''-(((1E,1'E,1''E)-benzene-1,3,5-triyltris(ethene-2,1-diyl))tris(benzene- 4,1- diyl))triacetic acid (52)
Triester 67 (454.8 mg, 0.77 mmol) and LiOH.1H2O (292.7 mg, 6.98 mmol) were dissolved in EtOH/H2O (1:9) (30 mL) and magnetically stirred at refluxed for 17 h. After the reaction mixture was cooled, the solvent was removed under reduced pressure, and the remaining solution was diluted with water (10 mL). After cooling to 0 oC, HCl (1M) was added to the mixture until the pH was adjusted to 3 and a grey precipitate resulted. The residue was collected by vacuum filtration and was recrystallised from o THF/H2O to afford a light brown solid (240.6 mg, 57 %, m.p. 100 – 108 C). 1 H NMR (400 MHz, CDCl3): 7.43 (d, J = 8 Hz, 6H, H4’/H8’), 7.39 (s, 3H, H2), 7.23 (d, J
= 7.6 Hz, 6H, H5’/H7’), 7.08 (d, J = 16.4 Hz, 3H, H2’), 6.99 (d, J = 16.4 Hz, 3H, H1’),
3.57 (s, 6H, H9’). 13 C NMR (100 MHz, CDCl3): 175.3 (C10’), 139.3 (C3’), 137.3 (C1), 135.3 (C6’), 130.5
(C5’/C7’), 129.7 (C2’), 129.1 (C1’), 127.5 (C4’/C8’), 124.7 (C2), 41.5 (C9’). IR (neat): 3026 (O-H), 1701 (C=O). + HR-MS (ESI): C36H31O6 [M + H] calcd 559.2121, found 559.2120.
10.3 Experimental procedures - P a r t I | 159
10.3.4.6 4-Vinylbenzenesulfonamide (74)
Sodium 4-vinylbenzenesulfonate (1g, 4.85 mmol) was added into a magnetically stirred solution of thionyl chloride (4.62 g, 38.8 mmol) at 0 oC, making sure that the solution did not exceed 10°C. This solution was further treated with dry DMF (5 mL). The reaction mixture was magnetically stirred for 6 h at room temperature and then stored at 4 oC for 15 h. The resulting mixture was subsequently poured into ice-water (15 mL). The solution was extracted with diethyl ether (3 x 20 mL) and the combined organic layers were washed with water (20 mL), dried (NaSO4) and filtered. The clear solution was reduced to half the volume and was syringed into an excess amount of anhydrous ammonia. The mixture was stirred for 1 h at below -50oC and was then warmed to room temperature with simultaneous evaporation for 15 h. To the dry mixture, a solution of
H2SO4 (7 mL, 50 %) was added and this solution was then extracted with CH2Cl2 (3 x
15 mL), dried (Na2SO4) and filtered. The solvent was removed under reduced pressure and the desired product was collected as a white solid (0.72 g, 81 %). The spectral data matched those reported in the literature.141 1 H NMR (400 MHz, CDCl3): 7.88 (d, J = 8.3 Hz, 2H, H2/H6), 7.53 (d, J = 8.3 Hz, 2H,
H3/H5), 6.75 (dd, J = 17.6 Hz, J = 10.7 Hz, 1H, H7), 5.89 (d, J = 17.6 Hz, 1H, H8), 5.43
(d, J = 10.7 Hz, 1H, H9), 4.80 (s, 2H, H10).
160 | Chapter 10: Experimentals
10.3.4.7 4-(1H-Tetrazol-5-yl)benzaldehyde (78)
4-Cyanobenzaldehyde (500 mg, 3.8 mmol), anhydrous ZnCl2 (779 mg, 5.72 mmol) and
NaN3 (371.8 mg, 5.72 mmol) were ground in a mortar and pestle under argon. The o mixture of solids were stirred and heated at 90 C for 15 h. 2M HCl (2 mL) in H2O (20 mL) was added to the reaction and the mixture was magnetically stirred at room temperature for a further 1 h. The resulting grey precipitate was washed with H2O (20 mL) and CH2Cl2 (20 mL) and the residue collected by vacuum filtration. The solid product was dried under high vacuum overnight as a fine white powder (152 mg, 23 %). The spectral data matched those reported in the literature.148 1 H NMR (400 MHz, CDCl3): 9.97 (s, 1H, H8), 8.17 (d, J = 6.8 Hz, 2H, H2/H4), 7.93 (d,
J = 6.8 Hz, 2H, H1/H5).
10.3.4.8 5-(4-vinylphenyl)-1H-tetrazole (79)
4-Vinylbenzonitrile (0.33 g, 2.55 mmol), sodium azide (0.49 g, 7.66 mmol) and triethylamine hydrochloride (1.05 g, 7.66 mmol) were dissolved in toluene (3 mL). The solution was stirred at 80 °C for 16 h and cooled to room temperature. The solution was extracted with water (3 x 5 mL). The combined aqueous layers were acidifed with 36 % HCl to give a white precipitate, which was collected via vacuum filtration as a white solid (0.36 g, 82 %, 195 – 202 °C) . 10.3 Experimental procedures - P a r t I | 161
1 H NMR (500 MHz, DMSO): δ 8.03 (d, J = 8.5 Hz, 2H, H2/H4), 7.71 (d, J = 8.5 Hz, 2H,
H1/H5), 6.82 (dd, J = 17.7, 11.0 Hz, 1H, H7), 6.00 (dd, J = 17.7, 0.7 Hz, 1H, H11), 5.41
(dd, J = 11.0, 0.7 Hz, 1H, H10). 13 C NMR (101 MHz, DMSO): δ 139.79 (C9), 135.74 (C8), 127.25 (C7), 127.05 (C2/C4),
127.04 (C1/C5), 123.29 (C3), 116.53 (C6). IR (neat): 2453, 2103, 1878. + HR-MS (AP+): C9H9N4 [M + H] calcd 173.0827, found 173.0826.
10.3.4.9 5-(4-vinylbenzyl)-1H-tetrazole (81)
2-(4-Vinylphenyl)acetonitrile (59) (0.40 g, 2.81 mmol), sodium azide (0.55 g, 8.44 mmol) and triethylamine hydrochloride (1.16 g, 8.44 mmol) were dissolved in toluene (3 mL). The solution was stirred at 80 °C for 16 h and cooled to room temperature. The solution was extracted with water (3 x 5 mL). The combined aqueous layers were acidifed with 36 % HCl to give a white precipitate, which was collected via vacuum filtration as a white solid (0.37 g, 71 %, 190 – 204 °C) . 1 H NMR (400 MHz, DMSO) δ 7.44 (d, J = 7.5 Hz, 2H, H2/H4), 7.25 (d, J = 7.7 Hz, 2H,
H1/H5), 6.71 (dd, J = 17.6, 10.9 Hz, 1H, H7), 5.81 (d, J = 17.7 Hz, 1H, H12), 5.24 (d, J =
10.9 Hz, 1H, H11), 4.28 (s, 2H, H9). 13 C NMR (101 MHz, DMSO): δ 154.78 (C10), 136.16 (C7), 135.92 (C3), 135.50 (C6),
128.92 (C2/C4), 126.44 (C1/C5), 114.28 (C8), 28.57 (C9). IR (neat): 2616, 2114, 1819, 1575 + HR-MS (AP+): C10H11N4 [M + H] calcd 187.0984, found 187.0980.
162 | Chapter 10: Experimentals
10.3.4.10 4,4',4''-((1E,1'E,1''E)-benzene-1,3,5-triyltris(ethene-2,1- diyl))tribenzonitrile (82)
Tribromobezene (200 mg, 0.63 mmol), Pd2(dba)3∙CHCl3 (94.5 mg, 0.095 mmol) and
[(t-Bu)3PH]BF4 (110 mg, 0.38 mmol) were dissolved in dry DMF. Cy2NMe (496 mg, 2.54 mmol) and 4-vinylbenzonitrile (270.8 mg, 2.09 mmol) were added and the reaction mixture was subjected a freeze-pump-thaw cycle three times. The resulting solution was stirred at 80 oC for 20 h. After cooling, a brown precipitate was collected and washed with methanol. The product is collected via vacuum filtration and dried under vacuum as an olive green powder (143 mg, 50%, m.p. 251 oC). 1 H NMR (500 MHz, DMSO-d6): 7.89 (s, 3H, H2), 7.88 (d, J = 8 Hz, 6H, H5’/H7’), 7.83
(d, J = 8 Hz, 6H, H4’/H8’), 7.54 (d, J = 16.5 Hz, 3H, H2’), 7.49 (d, J = 16.5 Hz, 3H, H1’). 13 C NMR (500 MHz, DMSO-d6): 141.7 (C3’), 137.4 (C10), 132.7 (C5’/C7’), 131.6 (C2’),
127.9 (C1’), 127.2 (C4’/C8’), 125.4 (C2), 118.9 (C9’), 109.7 (C6’). IR (neat): 2222 (C≡N), 1599, 1505. + HR-MS (API): C33H22N3 [M + H] calcd 460.1814, found 460.1814.
10.3 Experimental procedures - P a r t I | 163
10.3.4.11 2,2',2''-(4,4',4''-(1E,1'E,1''E)-2,2',2''-(benzene-1,3,5-triyl)tris(ethene-2,1- diyl)tris(benzene-4,1-diyl))triacetonitrile (83)
Tribromobenzene (0.5 g, 1.6 mmol), Pd2(dba)3∙CHCl3 (0.25 g, 0.24 mmol) and [(t-
Bu)3PH]BF4 (0.14 g, 0.4.8 mmol) were dissolved in dry DMF (4 mL). Cy2NMe (1.38 mL, 6 mmol) and 2-(4-vinylphenyl)acetonitrile (59) (0.76 g, 5 mmol) were added and the reaction mixture was subjected a freeze-pump-thaw cycle three times. The resultant solution was stirred at 80°C for 24 h. After cooling, water (4 mL) was added and the solution was extracted with CH2Cl2 (3 x 10 mL). The combined organic extracts were washed with water (3 x 10 mL) and brine (10 mL), dried with MgSO4 and filtered. The organic layer was concentrated under reduced pressure to give a yellow residue. The residue was subjected to column chromatography (5 % ethyl acetate/toluene) to afford a yellow solid which was then recrystallised (THF/hexane) (0.31 g, 39 %, 243 °C). 1 H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 8.0 Hz, 6H, H5’/H7’), 7.54 (s, 3H, H2), 7.34
(d, J = 8.0 Hz, 6H, H4’/H8’), 7.20 – 7.06 (m, 6H, H1’/H2’), 3.77 (s, 6H, H9’). 13 C NMR (101 MHz, CDCl3) δ 137.93 (C3’), 137.15 (C1), 129.08 (C6’), 128.49
(C1’/C2’), 128.47 (C5’/C7’), 127.32 (C4’/C8’), 124.36 (C2), 117.88 (C10’), 23.53 (C9’). IR (neat): 2247 (C≡N), 1586, 1511, 1416.
HR-MS (ES-): C36H27N3 [M - H] calcd 500.2121, found 500.2127.
164 | Chapter 10: Experimentals
10.3.4.12 1,3,5-tris(4-(1H-tetrazol-5-yl)styryl)benzene (75)
A mixture of 4,4',4''-((1E,1'E,1''E)-benzene-1,3,5-triyltris(ethene-2,1- diyl))tribenzonitrile (82) (0.1 g, 0.217 mmol), NH4Cl (0.41 g, 7.62 mol) and NaN3 (0.49 g, 7.62 mol) in DMF (8 mL) was stirred at 125 °C for 16 h. After cooling to r.t., the DMF was removed and the residue was suspended and stirred in 2M HCl (4 mL) for 30 min. The resulting light brown solid (0.118 g, 92 %, >300 °C) was filtered and dried under vacuum. 1 H NMR (500 MHz, DMSO) δ 8.08 (d, J = 8.4 Hz, 6H, H5’/H7’), 7.89 (s, 3H, H2), 7.88
(d, J = 6.2 Hz, 6H, H4’/H8’), 7.51 (d, J = 4.6 Hz, 6H, H1’/H2’). 13 C NMR (126 MHz, DMSO): δ 155.33 (C10’), 140.02 (C3’), 137.85 (C1), 132.92 (C9’),
130.28 (C2’), 128.46 (C1’), 127.64 (C5’/C7’), 127.62 (C4’/C8’), 123.15 (C2). IR (neat): 2729.60, 1607.70, 1495.98, 1065.09, 960.08.
HR-MS (ES-): C33H24N12 [M - H] calcd 587.2162, found 587.2169.
10.3 Experimental procedures - P a r t I | 165
10.3.4.13 1,3,5-tris(4-((1H-tetrazol-5-yl)methyl)styryl)benzene (76)
A mixture of 2,2',2''-(4,4',4''-(1E,1'E,1''E)-2,2',2''-(benzene-1,3,5-triyl)tris(ethene-2,1- diyl)tris(benzene-4,1-diyl))triacetonitrile (83) (0.14 g, 0.28 mmol), NH4Cl (0.52 g, 9.77 mol) and NaN3 (0.64 g, 9.77 mol) in DMF (3 mL) was stirred at 125 °C for 16 h. After cooling to r.t., the DMF was removed and the residue was suspended and stirred in 2M HCl (4 mL) for 30 min. The resulting light brown solid (0.128 g, 73 %, >300 °C) was filtered, washed with hot methanol and dried under vacuum.
H NMR (400 MHz, DMSO) δ 16.12 (s, 3H, H14’), 7.57 (d, J = 7.1 Hz, 6H, H4’/H8’ ),
7.35 (d, J = 16.3 Hz, 6H, H5’/H7’), 7.28 (s, 3H, H2), 7.24 (d, J = 17.1 Hz, 6H, H1’/H2’),
4.30 (s, 6H, C9’). 13 C NMR (400 MHz, DMSO-d6): 28.66 (C9’), 123.91 (C2), 126.82(C1’), 128.11 (C2’),
128.59 (C4’/C8’), 129.15 (C5’/C7’), 135.41 (C3’), 135.86 (C1), 137.78 (C6’), 157.19 (C10’). IR (neat): 3029, 1587, 1512, 1417. + HR-MS (ES+): C36H31N12 [M + H] calcd 631.2803, found 631.2795.
166 | Chapter 10: Experimentals
10.3.4.14 4,4',4''-((1E,1'E,1''E)-benzene-1,3,5-triyltris(ethene-2,1- diyl))tribenzamide (84)
A solution of 4,4',4''-((1E,1'E,1''E)-benzene-1,3,5-triyltris(ethene-2,1- diyl))tribenzonitrile (82) (100 mg, 0.2 mmol) was stirred at 75 °C in trifluoroacetic acid-
H2SO4 (4:1, v/v) (1 mL). After 16 h, the solution was quenched with iced water and the brown precipitate (112 mg, 100%, >300 °C) was collected under vacuum. 1H NMR was not obtained (insoluble compound). 13 C NMR (101 MHz, solid) δ 129.26 (s, C9’). IR (neat): 3188.19 (N-H), 1605.85 (C=O) + HR-MS (ES+): C33H28N3O3 [M + H] calcd 514.2098, found 514.2131.
10.3.4.15 2,2',2''-(((1E,1'E,1''E)-Benzene-1,3,5-triyltris(ethene-2,1- diyl))tris(benzene-4,1-diyl))triacetamide (85)
A solution of 2,2',2''-(((1E,1'E,1''E)-benzene-1,3,5-triyltris(ethene-2,1- diyl))tris(benzene-4,1-diyl))triacetonitrile (83) (100 mg, 0.19 mmol) was stirred at 75
°C in trifluoroacetic acid-H2SO4 (4:1, v/v) (1 mL). After 16 h, the solution was 10.3 Experimental procedures - P a r t I | 167 quenched with iced water and the brown precipitate (103.6 mg, 93 %, >300 °C) was collected under vacuum. 1H NMR was not obtained (insoluble compound). 13 C NMR (101 MHz, solid) δ 129.39 (s, C9’). IR (neat): 3188.73 (N-H), 1656.19 (C=O)
10.3.4.16 tert-Butyl(3,5-dibromophenoxy)dimethylsilane (100)
3, 5-Dibromophenol (500 mg, 1.98 mmol), 4-dimethylaminopyridine (24.2 mg, 0.19 mmol) and imidazole (216.2 mg, 3.17 mmol) were dissolved in CH2Cl2 (15 mL) and cooled to 0 °C. tert-Butyldimethylsilane chloride (329 mg, 2.18 mmol) was slowly added and the solution was warmed to room temperature and stirred for 18 h. After reaction is completed, the solution was filtered and solvent from the filtrate was removed under reduced pressure. The resultant yellow oil was dissolved in diethyl ether and acidified with 2 % HCl to pH 1. The organic layer was then washed with brine (3 x
15 mL), dried with Na2SO4 and filtered. The organic layer was concentrated under reduced pressure and was then purified by column chromatography (10 % ethyl acetate/hexanes) to afford a light yellow oil (603 mg, 83 %). The spectral data for this compound matched those reported in literature.449 1 H NMR (400 MHz, CDCl3): 7.25 (s, 2H, H2/H6), 6.92 (s, 1H, H4), 0.96 (s, 9H,
H9/H10/H11), 0.20 (s, 6H, H7/H8).
168 | Chapter 10: E xperimentals
10.3.4.17 Dimethyl 2,2'-(((1E,1'E)-(5-((tert-butyldimethylsilyl)oxy)-1,3- phenylene)bis(ethene-2,1-diyl))bis(4,1-phenylene))diacetate (102)
tert-butyl(3,5-dibromophenoxy)dimethylsilane (100) (500 mg, 1.36 mmol),
Pd2(dba)3∙CHCl3 (141.3 mg, 0.136 mmol) and [(t- Bu)3PH]BF4 (39.61 mg, 0.136 mmol) were dissolved in dry DMF (12 mL). Cy2NMe (0.87 mL, 4.09 mmol) and methyl 2-(4-vinylphenyl)acetate (54) (529.3 g, 3 mmol) were added and the reaction mixture was subjected a freeze-pump-thaw cycle three times. The resultant solution was stirred at 80 °C for 16 h. After cooling, water (10 mL) was added and the solution was extracted with CH2Cl2 (3 x 15 mL). The combined organic extracts were washed with water (3 x 15 mL) and brine (15 mL), dried with MgSO4 and filtered. The organic layer was concentrated under reduced pressure to give a yellow residue. The residue was subjected to column chromatography (10 % ethyl acetate/toluene) to afford a yellow oil (87.3 mg, 11 %). 1 H NMR (400 MHz, CDCl3): δ 7.49 (d, J = 7.7 Hz, 4H, H5’/H7’), 7.29 (d, J = 7.7 Hz, / 4H, H4’/H8’), 7.24 (t, 1H, H4), 7.13 – 7.01 (m, 4H, H1’ H2’), 6.89 (s, 2H, H2), 3.71 (s, 6H,
H11’), 3.65 (s, 4H, H9’), 1.03 (s, 9H, H10/ H11/H12), 0.26 (s, 6H, H7/H8). 13 C NMR (151 MHz, CDCl3) δ 172.08 (C10’), 156.45 (C1), 139.06 (C3’), 136.34 (C3),
133.55 (C6’), 129.77 (C5’/C7’), 128.76 (C2’), 128.60 (C1’), 126.91 (C4’/C8’), 118.52 (C4),
117.49 (C2), 52.26 (C11’), 41.10 (C9’), 29.85 (C9), 25.89 (C10/C11/C12), -4.13 (C7/C8). IR (neat): 2929, 2856, 1738 + HR-MS (ES+): C34H40O5Si [M + Na] calcd 579.2552, found 579.2543.
10.3 Experimental procedures - P a r t I | 169
10.3.4.18 Dimethyl 2,2'-(4,4'-(1E,1'E)-2,2'-(5-hydroxy-1,3-phenylene)bis(ethene- 2,1-diyl)bis(4,1-phenylene))diacetate (101)
3,5-dibromophenol (1 g, 3.96 mmol), Pd2(dba)3∙CHCl3 (205 mg, 0.19 mmol) and [(t-
Bu)3PH]BF4 (57.6 mg, 0.19 mmol) were dissolved in dry DMF (12 mL). Cy2NMe (2.12 mL, 9.92 mmol) and methyl 2-(4-vinylphenyl)acetate (54) (1.33 g, 8.73 mmol) were added and the reaction mixture was subjected a freeze-pump-thaw cycle three times. The resultant solution was stirred at 80°C for 24 h. After cooling, water (10 mL) was added and the solution was extracted with CH2Cl2 (3 x 15 mL). The combined organic extracts were washed with water (3 x 15 mL) and brine (15 mL), dried with MgSO4 and filtered. The organic layer was concentrated under reduced pressure to give a yellow residue. The residue was subjected to column chromatography (10 % ethyl acetate/toluene) to afford a yellow oil (1.57 g, 89 %) 1 H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.2 Hz, 4H, H5’/H7’), 7.28 (d, J = 8.1 Hz, / 4H, H4’/H8’), 7.24 (s, 1H, H4), 7.04 (q, J = 16.3 Hz, 4H, H1’ H2’), 6.86 (s, 2H, H2), 5.51
(s, 1H, OH), 3.72 (s, 6H, H11’), 3.65 (s, 4H, H9’). 13 C NMR (101 MHz, CDCl3) δ 172.33 (C10’), 156.42 (C1), 139.25 (C3’), 136.27 (C3),
133.51 (C6’), 129.77 (C5’/C7’), 128.95 (C2’), 128.35 (C1’), 126.93 (C4’/C8’), 118.18 (C4),
112.54 (C2), 52.34 (C11’), 40.08 (C9’). IR (neat): 2928, 2853, 1731. + HR-MS (ES+): C28H26O5 [M + Na] calcd 465.1696, found 465.1678.
170 | Chapter 10: Experimentals
10.3.4.19 Dimethyl 2,2'-(4,4'-(1E,1'E)-2,2'-(5-(trifluoromethylsulfonyloxy)-1,3- phenylene)bis(ethene-2,1-diyl)bis(4,1-phenylene))diacetate (99)
To a cooled solution of dimethyl 2,2'-(4,4'-(1E,1'E)-2,2'-(5-hydroxy-1,3- phenylene)bis(ethene-2,1-diyl)bis(4,1-phenylene))diacetate (101) (1.56 g, 3.52 mmol) and pyridine (0.56 mL, 7.05 mmol) dissolved in CH2Cl2 (10 mL) was added a solution of triflate anhydride (0.71 mL, 4.23 mmol) in CH2Cl2 (5 mL). The mixture was stirred at room temperature for 1 hour. The solution was diluted with diethyl ether (10 mL), quenched with 10 % HCl (5 mL) and washed with NaHCO3 and brine. It was then dried with MgSO4, filtered and concentrated under reduced pressure to give a yellow residue. The residue was subjected to column chromatography (2 % ethyl acetate/toluene) to afford a yellow solid (1.02 g, 51 %, m.p.) 1 H NMR (400 MHz, CDCl3) δ 7.60 (s, 1H, H4), 7.50 (d, J = 8.0 Hz, 4H, H5’/H7’), 7.31 / (d, J = 8.0 Hz, 4H, H4’/H8’), 7.27 (s, 2H, H2), 7.11 (dd, J = 37.4, 16.3 Hz, 4H, H1’ H2’),
3.72 (s, 6H, H11’), 3.64 (s, 4H, H9’). 19 F NMR (282 MHz, CDCl3) δ -73.20 (s). 13 C NMR (126 MHz, CDCl3) δ 171.95 (C10’), 150.53 (C1), 140.34 (C3’), 140.28 (C3),
135.50 (C6’), 134.35 (C2’), 131.08 (C1’), 129.91 (C5’/C7’), 127.15 (C4’/C8’), 126.62 (C2),
124.59 (C5), 117.55 (C4), 52.28 (C11’), 41.07 (C9’). IR (neat): 3030, 2954, 1724. + HR-MS (ES+): C29H25O7SF3 [M + Na] calcd 597.1180, found 597.1171.
10.3 Experimental procedures - P a r t I | 171
10.3.4.20 1-(Azidomethyl)-4-vinylbenzene (103)
4-Vinylbenzyl choride (2.0 g, 13 mmol) and sodium azide (1.71 g, 26 mmol) were dissolved in DMF (30 mL) and stirred at rt. After 24 h, water (20 mL) was added and the solution was extracted with CH2Cl2 (3 x 20 mL). The combined organic extracts was dried with Na2SO4 and filtered. The organic layer was concentrated under reduced pressure to give a yellow residue. The residue was subjected to column chromatography (10 % ethyl acetate/hexanes) to afford a bright yellow oil (1.83 g, 88 %). The spectral data for this compound matched those reported in literature.235 1 H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 8.0 Hz, 2H, H2/H4), 7.28 (d, J = 8.0 Hz, 2H,
H1/H5), 6.73 (dd, J = 17.6, 10.9 Hz, 1H, H7), 5.78 (d, J = 17.6 Hz, 1H, H12), 5.28 (d, J =
10.9 Hz, 1H, H11), 4.33 (s, 2H, H9).
10.3.4.21 Dimethyl 2,2'-(((1E,1'E)-(5-((4-(chloromethyl)benzyl)oxy)-1,3- phenylene)bis(ethene-2,1-diyl))bis(4,1-phenylene))diacetate (110)
Dimethyl 2,2'-(4,4'-(1E,1'E)-2,2'-(5-hydroxy-1,3-phenylene)bis(ethene-2,1-diyl)bis(4,1- phenylene))diacetate (101) (0.5 g, 1.13 mmol), α,α′-dichloro-p-xylene (0.24 g, 1.35 mmol) and K2CO3 (0.36 g, 2.4 mmol) were dissolved in acetone/DMF (4 mL, 5:1). The vessel was then subjected to three freeze-pump-thaw cycles. Potassium iodide (cat.) was added and the solution was heated to reflux for 36 h. After cooling, the acetone was removed under vacuum and the resultant solution was diluted with water and extracted 172 | C hapter 10: Experimentals with CH2Cl2 (3 x 5 mL). The organic layer was then washed with water (2 x 5 mL) and brine (2 x 5 mL), dried with MgSO4 and filtered. The solvent was removed under vacuum and the yellow residue was then subjected to column chromatography (20 % ethyl acetate/hexanes) to afford a yellow oil (204.3 mg, 31 %). 1 H NMR (600 MHz, CDCl3) δ 7.49 (d, J = 8.2 Hz, 2H, H14’/H16’), 7.47 (d, J = 8.3 Hz,
4H, H5’/H7’), 7.43 (d, J = 8.3 Hz, 2H, H13’/H17’), 7.30 (d, J = 8.3 Hz, 4H, H4’/H8’), 7.26
(s, 1H, H4), 7.12 (d, J = 16.3 Hz, 2H, H2’), 7.06 (d, J = 16.3 Hz, 2H, H1’), 7.04 (s, 2H,
H2), 5.11 (s, 2H, H12’), 4.60 (s, 2H, H19’), 3.72 (s, 6H, H11’), 3.65 (s, 4H, H9’). 13 C NMR (151 MHz, CDCl3) δ 171.91 (C10’), 159.26 (C1), 138.94 (C18’), 137.24 (C3’),
137.20 (C3), 136.04 (C15’), 133.49 (C6’), 129.64 (C5’/C7’), 128.86 (C14’/C16’), 128.84
(C2’), 128.30 (C1’), 127.78 (C13’/C17’), 126.77 (C4’/C8’), 118.33 (C4), 111.95 (C2), 69.58
(C12’), 52.08 (C11’), 45.94 (C19’), 40.87 (C9’). IR (neat): 3027, 2950, 1732, 1582, 1154. + HR-MS (EI+): C36H34O5Cl [M + H] calcd 581.2095, found 581.2089.
10.3.4.22 Dimethyl 2,2'-(((1E,1'E)-(5-((4-(azidomethyl)benzyl)oxy)-1,3- phenylene)bis(ethene-2,1-diyl))bis(4,1-phenylene))diacetate (114)
A solution of dimethyl 2,2'-(((1E,1'E)-(5-((4-(chloromethyl)benzyl)oxy)-1,3- phenylene)bis(ethene-2,1-diyl))bis(4,1-phenylene))diacetate (110) (150 mg, 0.25 mmol) and NaN3 (42 mg, 0.64 mmol) in DMF (2 mL) was gradually heated to 60 °C and stirred for 8 h. The cooled solution was diluted with water (5 mL) and extracted with diethyl ether (3 x 10 mL). The solution was then washed with water (2 x 5 mL) and brine (2 x 5 mL), dried with MgSO4 and filtered. The solvent was removed under vacuum. The yellow liquid was then subjected to preparative TLC (20 % ethyl acetate/hexanes) to afford a light yellow liquid product (98.3 mg, 65 %). 1 H NMR (500 MHz, CDCl3) δ 7.51 (d, J = 8.1 Hz, 2H, H14’/H16’), 7.49 (d, J = 8.2 Hz,
4H, H5’/H7’), 7.37 (d, J = 8.2 Hz, 2H, H13’/H17’), 7.29 (d, J = 8.2 Hz, 4H, H4’/H8’), 7.27 10.3 Experimental procedures - P a r t I | 173
(s, 1H, H4), 7.12 (d, J = 16.3 Hz, 2H, H2’), 7.07 (d, J = 16.3Hz, 2H, H1’), 7.05 (s, 2H,
H2), 5.15 (s, 2H, H12’), 4.37 (s, 2H, H19’), 3.72 (s, 6H, H11’), 3.65 (s, 4H, H9’). 13 C NMR (126 MHz, CDCl3) δ 172.01 (C10’), 159.43 (C1), 139.12 (C18’), 137.24 (C3’),
136.20 (C3), 135.30 (C15’), 133.63 (C6’), 129.76 (C5’/C7’), 129.03 (C2’), 128.61
(C14’/C16’), 128.46 (C1’), 128.07 (C13’/C17’), 126.90 (C4’/C8’), 118.47 (C4), 112.10 (C2),
69.83 (C12’), 54.62 (C19’), 52.22 (C11’), 41.05 (C9’). IR (neat): 3025, 2949, 2095 (N=N=N), 1730 + HR-MS (EI+): C36H34O5N3 [M + H] calcd 588.2512, found 588.2498.
10.3.4.23 2,2'-(((1E,1'E)-(5-((4-(azidomethyl)benzyl)oxy)-1,3-phenylene)bis(ethene- 2,1-diyl))bis(4,1-phenylene))diacetic acid (109)
Dimethyl 2,2'-(((1E,1'E)-(5-((4-(azidomethyl)benzyl)oxy)-1,3-phenylene)bis(ethene- 2,1-diyl))bis(4,1-phenylene))diacetate (114) (100 mg, 0.17 mmol) was dissolved in warm MeOH (0.5 mL). Following the addition of water (4.5 mL) and LiOH (28.6 mg, 0.68 mmol), the solution was heated at reflux for 16 h. After cooling to r.t., the MeOH was removed. The solution was then acidified with 2M HCl to pH ~2 for a light yellow precipitate. The solid (83 mg, 87 %, 165 – 177 °C) was collected and dried under vacuum. 1 H NMR (400 MHz, CD3OD, 50 °C) δ 7.46 (d, J = 7.8 Hz, 2H, H14’/H16’), 7.45 (d, J =
8.1 Hz, 4H, H5’/H7’), 7.33 (d, J = 8.1 Hz, 2H, H13’/H17’), 7.24 (d, J = 8.2 Hz, 4H,
H4’/H8’), 7.22 (s, 1H, H4), 7.10 (d, J = 16.4 Hz, 2H, H2’), 7.03 (d, J = 16.5 Hz, 2H, H1’),
7.01 (s, 2H, H2), 5.08 (s, 2H, H12’), 4.32 (s, 2H, H19’), 3.58 (s, 4H, H9’). 13 C NMR (126 MHz, MeOD) δ 175.55 (C10’), 160.70 (C1), 140.41 (C18’), 138.81 (C3’),
137.36 (C3), 136.67 (C15’), 135.49 (C6’), 130.75 (C5’/C7’), 129.88 (C2’), 129.57
(C14’/C16’), 129.31 (C1’), 129.01 (C13’/C17’), 127.76 (C4’/C8’), 119.35 (C4), 113.00 (C2),
70.64 (C12’), 55.28 (C19’), 41.72 (C9’). 174 | Chapter 10: Experimentals
IR (neat): 3033, 2096 (N=N=N), 1721, 1575, 1415. + HR-MS (EI+): C34H30O5N3 [M + H] calcd 560.2205, found 560.2185.
10.3.5 Confocal microscopy 135C-treated and untreated S. aureus NCTC6571 were washed, resuspended and diluted to 0.5 McFarland in 0.85 % saline. Bacteria were heat-fixed onto glass coverslips and if appropriate, the sample was stained with a 10mM methanol solution of F1-DIBO. Samples were covered with a glycerol mounting media, then with a microscope slide, before sealing with an organic polymer. The samples were observed with a Nikon Ti-E inverted motorised microscope with a Nikon A1Si spectral detector confocal system.
10.4 Experimental procedures- Part II 10.4.1 General procedure for Mizoroki–Heck cross-coupling reactions An oven-dried glass vial was charged sequentially with the corresponding aryl triflate, nickel catalyst, phosphine ligand and base. The mixture is dissolved in toluene (3 mL), followed by the addition of butyl vinyl ether (206). The reaction vessel was sealed and stirred at 100 °C in a preheated oil bath. After 18 h, the reaction was cooled to RT, treated with 6 M HCl (1 mL), and stirred at RT for 1 h. The mixture is then extracted with Et2O (3 x 3 mL), and the combined organic layers were then dried (MgSO4), filtered and fused onto silica gel. Purification by flash column chromatography on silica produced the desired ketone products.
10.4.1.1 4-Acetylbenzonitrile (207)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate
(125.6 mg, 0.5 mmol), Ni[P(OPh)3]2(dppf) (30.8 mg, 0.025 mmol, 5 mol%), DiPrPF
(10.5 mg, 0.025 mmol, 5 mol%), Cy2NMe (107 µL, 0.5 mmol, 1.00 equiv.), butyl vinyl ether (67 µL, 0.5 mmol, 1.00 equiv.) and toluene (2 mL), the desired ketone (207) was isolated after treatment with acid and purification by flash column chromatography (1 % 10.4 Experimental procedures - P a r t I I | 175
EtOAc/hexanes) as a pale yellow solid (24 mg, 33 %). The spectral data were in accordance with those reported in literature.114 1 H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 8.1 Hz, 2H, 2 x Ar-H), 7.78 (d, J = 8.0 Hz,
2H, 2 x Ar-H), 2.65 (s, 3H, CH3).
10.4.1.2 1-(Naphthalen-2-yl)ethanone (230)
Following the general procedure, using naphthalen-2-yl trifluoromethanesulfonate (100 mg, 0.36 mmol), Ni[P(OPh)3]2(dppf) (22.3 mg, 0.018 mmol, 5 mol%), DiPrPF (7.5 mg,
0.018 mmol, 5 mol%), Cy2NMe (77 µL, 0.36 mmol, 1.00 equiv.), butyl vinyl ether (48 µL, 0.36 mmol, 1.00 equiv.) and toluene (2 mL), the desired ketone (230) was isolated after treatment with acid and purification by flash column chromatography (5 % EtOAc/hexanes) as a yellow solid (47.7 mg, 77.4 %). The spectral data were in accordance with those reported in literature.450 1 H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H, Ar-H), 8.04 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H), 7.97 (d, J = 8.1 Hz, 1H, Ar-H), 7.92 – 7.85 (m, 2H, 2 x Ar-H), 7.58 (m, 2H, 2 x Ar-H),
7.26 (s, 1H), 2.73 (s, 3H, CH3).
10.4.1.3 1-(Naphthalen-1-yl)ethanone (231)
Following the general procedure, using naphthalen-1-yl trifluoromethanesulfonate
(138.1 mg, 0.5 mmol), Ni[P(OPh)3]2(dppf) (30.8 mg, 0.025 mmol, 5 mol%), DiPrPF
(10.5 mg, 0.025 mmol, 5 mol%), Cy2NMe (107 µL, 0.5 mmol, 1.00 equiv.), butyl vinyl ether (67 µL, 0.5 mmol, 1.00 equiv.) and toluene (2 mL), the desired ketone (231) was isolated after treatment with acid and purification by flash column chromatography (5 % EtOAc/hexanes) as a yellow solid (25 mg, 56 %). The spectral data were in accordance with those reported in literature.451 176 | Chapter 10: Experimentals
1 H NMR (500 MHz, CDCl3) δ 8.75 (dd, J = 8.6, 1.0 Hz, 1H, Ar-H), 8.04 – 7.82 (m, 3H,
3 x Ar-H), 7.61 – 7.45 (m, 3H, 3 x Ar-H), 2.75 (s, 3H, CH3).
10.4 Experimental procedures - P a r t I I | 177
10.4.2 General procedure for Suzuki–Miyaura cross-coupling reactions An oven-dried glass vial was charged sequentially with the corresponding aryl boronic acid, aryl tosylate, nickel catalyst, phosphine ligand and base. The mixture was dissolved in 1,4-dioxane (3 mL) and the reaction vessel was sealed and stirred at 100 °C in a preheated oil bath. After 16 h, the reaction was cooled to RT, and the mixture was diluted with NaCl (2 mL) and extracted with CH2Cl2 (3 x 3 mL). The combined organic layers were then dried (Mg2SO4), filtered and fused onto silica gel. Purification by flash column chromatography on silica produced the desired aryl products.
10.4.2.1 [1,1'-Biphenyl]-4-carbonitrile (249)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (163.83 mg, 0.6 mmol), phenylboronic acid (106.82 mg, 0.6 mmol, 1.00 equiv.),
Ni[P(O-p-Tol)3]2(dppf) (23.72 mg, 0.018 mmol, 3 mol%), DPPB (7.67 mg, 0.018 mmol, 3 mol%), K3PO4 (254.6 mg, 1.2 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (249) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a white solid (89.8 mg, 76 %). The spectral data were in accordance with those reported in literature.452 1 H NMR (600 MHz, CDCl3) δ 7.70 (dd, J = 24.4, 8.4 Hz, 4H, 4 x Ar-H), 7.59 (d, J = 7.1 Hz, 2H, 2 x Ar-H), 7.49 (t, J = 7.5 Hz, 2H, 2 x Ar-H), 7.43 (t, J = 7.3 Hz, 1H, Ar-H). 13 C NMR (126 MHz, CDCl3) δ 145.76 (Ar-C), 139.26 (Ar-C), 132.69 (Ar-CH), 129.22 (Ar-CH), 128.77 (Ar-CH), 127.83 (Ar-CH), 127.33 (Ar-CH), 119.05 (C≡N), 111.01 (Ar-C).
10.4.2.2 1,1'-Biphenyl (252)
Following the general procedure, using phenyl 4-methylbenzenesulfonate (124.15 mg, 0.5 mmol), phenylboronic acid (154.2 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-
Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%), 178 | Chapter 10: Experimentals
K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (252) was isolated after a DCM workup and purification by flash column chromatography (100 % hexanes) as a white crystals (53.4 mg, 69 %). The spectral data were in accordance with those reported in literature.453 1 H NMR (500 MHz, CDCl3) δ 7.63 (dd, J = 8.0, 0.8 Hz, 4H, 4 x Ar-H), 7.47 (t, J = 7.7 Hz, 4H, 4 x Ar-H), 7.38 (t, J = 7.4 Hz, 2H, 2 x Ar-H). 13 C NMR (126 MHz, CDCl3) δ 141.39 (Ar-C), 128.89 (Ar-CH), 127.39 (Ar-C), 127.31 (Ar-CH).
10.4.2.3 2,6-Dimethoxy-1,1'-biphenyl (255)
Following the general procedure, using 2,6-dimethoxyphenyl 4-methylbenzenesulfonate (154.2 mg, 0.5 mmol), phenylboronic acid (154.2 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O- p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (255) was isolated after a DCM workup and purification by flash column chromatography (2 % EtOAc/hexanes) as colourless crystals (42.3 mg, 40 %). The spectral data were in accordance with those reported in literature.454 1 H NMR (500 MHz, CDCl3) δ 7.40 – 7.23 (m, 6H, 6 x Ar-H), 6.64 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 3.71 (s, 6H, CH3). 13 C NMR (126 MHz, CDCl3) δ 157.83 (Ar-C), 134.27 (Ar-CH), 131.03 (Ar-CH), 128.77 (s), 127.81 (Ar-CH), 126.91 (Ar-CH), 119.74 (Ar-C), 104.37 (Ar-CH), 56.07
(CH3).
10.4.2.4 3-(Trifluoromethyl)-1,1'-biphenyl (256)
Following the general procedure, using 3-(trifluoromethyl)phenyl 4- methylbenzenesulfonate (158.15 mg, 0.5 mmol), phenylboronic acid (154.2 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB 10.4 Experimental procedures - P a r t I I | 179
(6.39 mg, 0.015 mmol, 3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4- dioxane (3 mL), the desired aryl product (256) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a yellow oil (48.3 mg, 43 %). The spectral data were in accordance with those reported in literature.455 1 H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H, Ar-H), 7.79 (d, J = 7.6 Hz, 1H, Ar-H), 7.62 (d, J = 6.6 Hz, 3H, 3 x Ar-H), 7.57 (t, J = 7.7 Hz, 1H, Ar-H), 7.50 (t, J = 7.2 Hz, 2H, 2 x Ar-H), 7.43 (t, J = 7.3 Hz, 1H, Ar-H). 13 C NMR (126 MHz, CDCl3) δ 142.18 (Ar-C), 139.91 (Ar-C), 131.32 (q, J = 32.2 Hz, Ar-CH), 130.56 (Ar-CH), 129.37 (Ar-CH), 129.14 (Ar-CH), 128.90 (Ar-CH), 128.18
(Ar-CH), 127.33 (Ar-CH), 124.15 – 124.08 (m, CF3), 124.06 (Ar-C). 19 F NMR (471 MHz, CDCl3) δ -62.54 (s, CF3).
10.4.2.5 1,1':4',1''-Terphenyl (257)
Following the general procedure, using [1,1'-biphenyl]-4-yl 4-methylbenzenesulfonate (162.2 mg, 0.5 mmol), phenylboronic acid (89 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-
Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%),
K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (257) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a white solid (49.3 mg, 43 %). The spectral data were in accordance with those reported in literature.456 1H NMR (500 MHz, CDCl3) δ 7.74 – 7.61 (m, 8H, 8 x Ar-H), 7.47 (t, J = 7.5 Hz, 4H, 4 x Ar-H), 7.37 (t, J = 7.3 Hz, 2H, 2 x Ar-H). 13 C NMR (126 MHz, CDCl3) δ 140.87 (Ar-C), 140.28 (Ar-C), 128.97 (Ar-CH), 127.65 (Ar-CH), 127.49 (Ar-CH), 127.21 (Ar-CH).
180 | Chapter 10: Experimentals
10.4.2.6 4-(tert-Butyl)-1,1'-biphenyl (258)
Following the general procedure, using 4-(tert-butyl)phenyl 4-methylbenzenesulfonate (152.2 mg, 0.5 mmol), phenylboronic acid (154.2 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O- p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (258) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as gray crystals (62.5 mg, 59 %). The spectral data were in accordance with those reported in literature.457 1 H NMR (500 MHz, CDCl3) δ 7.65 – 7.57 (m, 2H, 2 x Ar-H), 7.58 – 7.52 (m, 2H, 2 x
Ar-H), 7.51 – 7.41 (m, 4H, 4 x Ar-H), 7.38 – 7.30 (m, 1H, Ar-H), 1.39 (s, 1H, CH3). 13 C NMR (126 MHz, CDCl3) δ 150.40 (Ar-C), 141.22 (Ar-C), 138.47 (Ar-C), 128.84
(Ar-CH), 127.17 (Ar-CH), 126.93 (Ar-CH), 125.86 (Ar-CH), 34.68 (C(CH3)3), 31.52
(C(CH3)3).
10.4.2.7 4-Methyl-1,1'-biphenyl (260)
Following the general procedure, using phenyl p-tolyl 4-methylbenzenesulfonate (121.1 mg, 0.5 mmol), phenylboronic acid (154.2 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-
Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%),
K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (260) was isolated after a DCM workup and purification by flash column chromatography (100 % hexanes) as white crystals (44.3 mg, 53 %). The spectral data were in accordance with those reported in literature.458 1 H NMR (500 MHz, CDCl3) δ 7.61 (d, J = 7.2 Hz, 2H, 2 x Ar-H), 7.53 (d, J = 8.1 Hz, 2H, 2 x Ar-H), 7.46 (t, J = 7.7 Hz, 2H, 2 x Ar-H), 7.35 (t, J = 7.4 Hz, 1H, 2 x Ar-H),
7.30 – 7.24 (d, J = 7.9 Hz, 2H, 2 x Ar-H), 2.43 (s, 3H, CH3). 10.4 Experimental procedures - P a r t I I | 181
13 C NMR (126 MHz, CDCl3) δ 141.30 (Ar-C), 138.50 (Ar-C), 137.15 (Ar-C), 129.62 (Ar-CH), 128.85 (Ar-CH), 127.31 (Ar-C), 127.13 (Ar-CH), 127.11 (Ar-CH), 21.23
(CH3).
10.4.2.8 2-Methyl-1,1'-biphenyl (261)
Following the general procedure, using o-tolyl 4-methylbenzenesulfonate (121.1 mg, 0.5 mmol), phenylboronic acid (154.2 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-
Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%),
K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (261) was isolated after a DCM workup and purification by flash column chromatography (100 % hexanes) as a colourless oil (26 mg, 31 %). The spectral data were in accordance with those reported in literature.453 1 H NMR (500 MHz, CDCl3) δ 7.49 – 7.44 (m, 2H, 2 x Ar-H), 7.42 – 7.36 (m, 3H, 3 x
Ar-H), 7.35 – 7.28 (m, 4H, 4 x Ar-H), 2.34 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 142.10 (Ar-C), 142.07 (Ar-C), 135.47 (Ar-C), 130.43 (Ar-CH), 129.93 (Ar-CH), 129.32 (Ar-CH), 128.19 (Ar-CH), 127.38 (Ar-CH), 126.89
(Ar-CH), 125.89 (Ar-CH), 20.60 (CH3).
10.4.2.9 1,1':3',1''-Terphenyl (263)
Following the general procedure, using 3-bromophenyl 4-methylbenzenesulfonate (163.6 mg, 0.5 mmol), phenylboronic acid (154.2 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O- p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (263) was isolated after a DCM workup and purification by flash column chromatography (100 % hexanes) as a white solid (52 mg, 45 %). The spectral data were in accordance with those reported in literature.459 182 | Chapter 10: Experimentals
1 H NMR (500 MHz, CDCl3) δ 7.86 (s, 1H, Ar-H), 7.70 (d, J = 7.7 Hz, 4H, 4 x Ar-H), 7.63 (d, J = 8.0 Hz, 2H, 2 x Ar-H), 7.58 – 7.53 (m, 1H, Ar-H), 7.51 (t, J = 7.6 Hz, 4H, 4 x Ar-H), 7.42 (t, J = 7.3 Hz, 2H, 2 x Ar-H). 13 C NMR (126 MHz, CDCl3) δ 141.92 (Ar-C), 141.32 (Ar-C), 129.32 (Ar-CH), 128.93 (Ar-CH), 127.53 (Ar-CH), 127.39 (Ar-CH), 126.29 (Ar-CH), 126.26 (Ar-CH).
10.4.2.10 [1,1':4',1''-Terphenyl]-4-carbonitrile (272)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), [1,1'-biphenyl]-4-ylboronic acid (99 mg, 0.5 mmol, 1.00 equiv.),
Ni[P(O-p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol,
3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (272) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a white solid (91.3 mg, 72 %). The spectral data were in accordance with those reported in literature.460 1 H NMR (500 MHz, CDCl3) δ 7.77-7.61 (m, 10H, 10 x Ar-H), 7.48 (t, J = 7.6 Hz, 2H, 2 x Ar-H), 7.39 (t, J = 7.4 Hz, 1H, Ar-H). 13 C NMR (126 MHz, CDCl3) δ 145.30 (Ar-C), 141.72 (Ar-C), 140.34 (Ar-C), 138.10 (Ar-C), 132.80 (Ar-CH), 129.07 (Ar-CH), 127.96 (Ar-CH), 127.89 (Ar-CH), 127.75 (Ar-CH), 127.72 (Ar-CH), 127.22 (Ar-CH), 119.10 (C≡N), 111.08 (Ar-C).
10.4.2.11 4'-Methoxy-[1,1'-biphenyl]-4-carbonitrile (273)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), (4-methoxyphenyl)boronic acid (76 mg, 0.5 mmol, 1.00 equiv.),
Ni[P(O-p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol,
3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (273) was isolated after a DCM workup and purification by flash column 10.4 Experimental procedures - P a r t I I | 183 chromatography (5 % EtOAc/hexanes) as a white solid (73.9 mg, 71 %). The spectral data were in accordance with those reported in literature.461 1 H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 8.5 Hz, 2H, 2 x Ar-H), 7.64 (d, J = 8.5 Hz, 2H, 2 x Ar-H), 7.54 (d, J = 8.8 Hz, 2H, 2 x Ar-H), 7.01 (d, J = 8.8 Hz, 2H, 2 x Ar-H),
3.86 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 160.33 (Ar-C), 145.32 (Ar-C), 132.67 (Ar-CH), 131.60 (Ar-C), 128.47 (Ar-CH), 127.21 (Ar-CH), 119.21 (C≡N), 114.67 (Ar-CH), 110.21 (Ar-
C), 55.51 (OCH3).
10.4.2.12 Methyl 4'-cyano-[1,1'-biphenyl]-4-carboxylate (274)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), (4-(methoxycarbonyl)phenyl)boronic acid (90 mg, 0.5 mmol,
1.00 equiv.), Ni[P(O-p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg,
0.015 mmol, 3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (274) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a white solid (79.9 mg, 67 %). The spectral data were in accordance with those reported in literature.462 1H NMR (500 MHz, CDCl3) δ 8.14 (d, J = 8.5 Hz, 2H, 2 x Ar-H), 7.75 (d, J = 8.5 Hz, 2H, 2 x Ar-H), 7.71 (d, J = 8.5 Hz, 2H, 2 x Ar-H), 7.65 (d, J = 8.4 Hz, 2H, 2 x Ar-H),
3.95 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 166.71 (C=O), 144.54 (Ar-C), 143.53 (Ar-C), 132.84 (Ar-CH), 130.47 (Ar-CH), 130.33 (Ar-C), 128.05 (Ar-CH), 127.36 (Ar-CH), 118.78
(C≡N), 111.95 (Ar-C), 52.42 (CH3).
184 | Chapter 10: Experimentals
10.4.2.13 4'-(Trifluoromethyl)-[1,1'-biphenyl]-4-carbonitrile (275)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), (4-(trifluoromethyl)phenyl)boronic acid (95 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg,
0.015 mmol, 3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (275) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a white solid (92.2 mg, 75 %). The spectral data were in accordance with those reported in literature.461 1 H NMR (500 MHz, CDCl3) δ 7.80 – 7.64 (m, 8H). 13 C NMR (126 MHz, CDCl3) δ 144.22 (Ar-C ), 142.77 (Ar-C), 132.89 (Ar-CH), 128.06
(Ar-CH), 127.73 (Ar-CH), 126.18 (Ar-C), 126.15 (Ar-C), 122.15 (CF3), 118.69 (C≡N), 112.07 (Ar-C).
10.4.2.14 4'-Fluoro-[1,1'-biphenyl]-4-carbonitrile (276)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), (4-fluorophenyl)boronic acid (70 mg, 0.5 mmol, 1.00 equiv.),
Ni[P(O-p-Tol)3]2(dppf) (19.8 mg, 0.015 immol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%), K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (276) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a white solid (71 mg, 72 %). The spectral data were in accordance with those reported in literature.463 1 H NMR (500 MHz, CDCl3) δ 7.71 (d, J = 8.6 Hz, 2H, 2 x Ar-H), 7.64 (d, J = 8.6 Hz, 2H, 2 x Ar-H), 7.56 (dd, J = 8.9, 5.2 Hz, 2H, 2 x Ar-H), 7.17 (t, J = 8.7 Hz, 2H, 2 x Ar- H). 13 C NMR (126 MHz, CDCl3) δ 163.29 (d, JC-F = 248.9 Hz, Ar-CF), 144.70 (Ar-C),
135.39 (d, JC-F = 3.2 Hz, Ar-C), 132.74 (Ar-CH), 129.06 (d, JC-F = 8.3 Hz, Ar-CH),
127.67 (Ar-CH), 118.92 (C≡N), 116.21 (d, JC-F = 21.7 Hz, Ar-CH), 111.06 (Ar-C). 10.4 Experimental procedures - P a r t I I | 185
10.4.2.15 2'-Methyl-[1,1'-biphenyl]-4-carbonitrile (277)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), o-tolylboronic acid (68 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-
Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%),
K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (277) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a light yellow solid (76 mg, 79 %). The spectral data were in accordance with those reported in literature.464 1 H NMR (500 MHz, CDCl3) δ 7.66 (d, J = 8.2 Hz, 2H, 2 x Ar-H), 7.39 (d, J = 8.2 Hz, 2H, 2 x Ar-H), 7.31 – 7.19 (m, 3H, 3 x Ar-H), 7.15 (d, J = 7.4 Hz, 1H, Ar-H), 2.22 (s,
3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 146.83 (Ar-C), 140.04 (Ar-C), 135.07 (Ar-C), 132.02 (Ar-CH), 130.73 (Ar-CH), 130.05 (Ar-CH), 129.47 (Ar-CH), 128.36 (Ar-CH), 126.16
(Ar-CH), 119.00 (C≡N), 110.77 (Ar-C), 20.37 (CH3).
10.4.2.16 4'-Methyl-[1,1'-biphenyl]-4-carbonitrile (278)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), p-tolylboronic acid (68 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-
Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%),
K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (278) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a light yellow solid (73.7 mg, 76 %). The spectral data were in accordance with those reported in literature.465 1 H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.4 Hz, 2H, 2 x Ar-H), 7.67 (d, J = 8.4 Hz, 2H, 2 x Ar-H), 7.49 (d, J = 8.1 Hz, 2H, 2 x Ar-H), 7.29 (d, J = 8.0 Hz, 2H, 2 x Ar-H),
2.42 (s, 3H, CH3). 186 | Chapter 10: Experimentals
13 C NMR (126 MHz, CDCl3) δ 145.69 (Ar-C), 138.85 (Ar-C), 136.35 (Ar-C), 132.66 (Ar-CH), 129.94 (Ar-CH), 127.55 (Ar-CH), 127.15 (Ar-CH), 119.14 (C≡N), 110.63
(Ar-C), 21.28 (CH3).
10.4.2.17 3'-Methyl-[1,1'-biphenyl]-4-carbonitrile (279)
Following the general procedure, using 4-cyanophenyl 4-methylbenzenesulfonate (136.5 mg, 0.5 mmol), m-tolylboronic acid (68 mg, 0.5 mmol, 1.00 equiv.), Ni[P(O-p-
Tol)3]2(dppf) (19.8 mg, 0.015 mmol, 3 mol%), DPPB (6.39 mg, 0.015 mmol, 3 mol%),
K3PO4 (212.1 mg, 1 mmol, 2.00 equiv.) and 1,4-dioxane (3 mL), the desired aryl product (279) was isolated after a DCM workup and purification by flash column chromatography (5 % EtOAc/hexanes) as a light yellow oil (61.7 mg, 64 %). The spectral data were in accordance with those reported in literature.466 1 H NMR (500 MHz, CDCl3) δ 7.61-7.56 (m, 4H, 4 x Ar-H), 7.29 (t, J = 7.5 Hz, 3H, 3 x
Ar-H), 7.15 (m, 1H, Ar-H), 2.34 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 145.84 (Ar-C), 139.18 (Ar-C), 138.85 (Ar-C), 132.57 (Ar-CH), 129.47 (Ar-CH), 129.07 (Ar-CH), 128.01 (Ar-CH), 127.76 (Ar-CH), 124.38
(Ar-CH), 119.04 (C≡N), 110.81 (Ar-C), 21.56 (CH3).
10.4.3 General procedure for the synthesis of aryl tosylates
The appropriate phenol was first dissolved in CH2Cl2 (15 mL), followed by the addition of triethylamine (1.1 equiv.). p-Tosyl chloride (1.1 equiv.) was then added with care and the mixture stirred at RT for 8 h. After complete conversion, water was added, and the reaction mixture was extracted with CH2Cl2 (2 x 10 mL). The organic layers were combined, dried (MgSO4), concentrated under reduced pressure, and recrystallised
(CH2Cl2) to give the desired product.
10.4 Experimental procedures - P a r t I I | 187
10.4.3.1 Phenyl 4-methylbenzenesulfonate (282)
Following the general procedure, using phenol (0.94 g, 10 mmol), p-tosyl chloride
(2.097 g, 11 mmol, 1.1 equiv.), Et3N (1.53 mL, 11 mmol, 1.1 equiv.) and CH2Cl2 (15 mL), the desired aryl tosylate (282) was isolated after a DCM workup and recrystallisation (CH2Cl2) as an off white solid (1.53 g, 61 %). The spectral data were in accordance with those reported in literature.467 1 H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.37 – 7.19 (m, 5H, 5 x Ar-H), 7.07 – 6.90 (m, 2H, 2 x Ar-H), 2.44 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 149.79 (Ar-C), 145.45 (Ar-C), 132.55 (Ar-C), 129.86 (Ar-CH), 129.72 (Ar-CH), 128.64 (Ar-CH), 127.20 (Ar-CH), 122.52 (Ar-CH), 21.84
(Ar-CH3).
10.4.3.2 2,6-Dimethoxyphenyl 4-methylbenzenesulfonate (283)
Following the general procedure, using 2,6-dimethoxyphenol (1.54 g, 0.01 mol), p-tosyl chloride (2.1 g, 0.011mol, 1.1 equiv.), Et3N (1.53 mL, 0.011 mol, 1.1 equiv.) and
CH2Cl2 (15 mL), the desired aryl tosylate (283) was isolated after a DCM workup and recrystallisation (CH2Cl2) as a light brown powder (1.79 g, 58 %). The spectral data were in accordance with those reported in literature.468 1 H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.33 (d, J = 8.2 Hz, 2H, 2 x Ar-H), 7.13 (t, J = 8.4 Hz, 1H, Ar-H), 6.55 (d, J = 8.5 Hz, 2H, 2 x Ar-H), 3.67
(s, 6H, , 2 x O-CH3), 2.46 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 153.67 (Ar-C), 144.54 (Ar-C), 135.14 (Ar-C), 129.27 (Ar-CH), 128.54 (Ar-C), 128.42 (Ar-CH), 127.48 (Ar-CH), 105.06 (Ar-CH), 56.07
(OCH3), 21.79 (Ar-CH3).
188 | Chapter 10: Experimentals
10.4.3.3 p-Tolyl 4-methylbenzenesulfonate (284)
Following the general procedure, using p-cresol (1.08 g, 10 mmol), p-tosyl chloride
(2.097 g, 11 mmol, 1.1 equiv.), Et3N (1.53 mL, 11 mmol, 1.1 equiv.) and CH2Cl2 (15 mL), the desired aryl tosylate (284) was isolated after a DCM workup and recrystallisation (CH2Cl2) as an off white solid (1.95 g, 75 %). The spectral data were in accordance with those reported in literature.469 1 H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.30 (d, J = 8.1 Hz, 2H, 2 x Ar-H), 7.06 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 6.85 (d, J = 8.5 Hz, 2H, 2 x Ar-H),
2.44 (s, 3H, CH3), 2.30 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 147.61 (Ar-C), 145.33 (Ar-C), 137.06 (Ar-C), 132.64 (Ar-C), 130.20 (Ar-CH), 129.82 (Ar-CH), 128.67 (Ar-CH), 122.20 (Ar-CH), 21.84
(CH3), 21.01 (CH3).
10.4.3.4 o-Tolyl 4-methylbenzenesulfonate (285)
Following the general procedure, using o-cresol (1.08 g, 10 mmol), p-tosyl chloride
(2.097 g, 11 mmol, 1.1 equiv.), Et3N (1.53 mL, 11 mmol, 1.1 equiv.) and CH2Cl2 (15 mL), the desired aryl tosylate (285) was isolated after a DCM workup and recrystallisation (CH2Cl2) as a pink solid (1.57 g, 60 %). The spectral data were in accordance with those reported in literature.468 1 H NMR (500 MHz, CDCl3) δ 7.74 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.32 (d, J = 8.1 Hz, 2H, 2 x Ar-H), 7.20 – 7.06 (m, 3H, 3 x Ar-H), 6.99 (d, J = 7.3 Hz, 1H, Ar-H), 2.45 (s,
3H, CH3), 2.08 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 148.47 (Ar-C), 145.43 (Ar-C), 133.32 (Ar-C), 131.73 (Ar-CH), 131.69 (Ar-CH), 129.91 (Ar-CH), 128.52 (Ar-CH), 127.08 (Ar-C), 127.01
(Ar-CH), 122.42 (Ar-CH), 21.83 (Ar-CH3), 16.41 (Ar-CH3).
10.4 Experimental procedures - P a r t I I | 189
10.4.3.5 4-(tert-Butyl)phenyl 4-methylbenzenesulfonate (286)
Following the general procedure, using 4-(tert-butyl)phenol (1.5 g, 10 mmol), p-tosyl chloride (2.097 g, 11 mmol, 1.1 equiv.), Et3N (1.53 mL, 11 mmol, 1.1 equiv.) and
CH2Cl2 (15 mL), the desired aryl tosylate (286) was isolated after a DCM workup and recrystallisation (CH2Cl2) as an off white solid (1.63 g, 54 %). The spectral data were in accordance with those reported in literature. 470 1 H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.28 (d, J = 8.0 Hz, 2H, 2 x Ar-H), 7.24 (d, J = 6.0 Hz, 2H, 2 x Ar-H), 6.86 (d, J = 8.8 Hz, 2H, 2 x Ar-H),
2.42 (s, 3H, CH3), 1.25 (s, 9H, 3 x CH3). 13 C NMR (126 MHz, CDCl3) δ 150.22 (Ar-C), 147.45 (Ar-C), 145.29 (Ar-C), 132.89 (Ar-C), 129.84 (Ar-CH), 128.64 (Ar-CH), 126.63 (Ar-CH), 121.82 (Ar-CH), 34.68
(C(CH3)3), 31.46 (C(CH3)3), 21.86 (Ar-CH3).
10.4.3.6 3-(Trifluoromethyl)phenyl 4-methylbenzenesulfonate (287)
Following the general procedure, using 3-(trifluoromethyl)phenol (1.21 mL, 0.01 mol), p-tosyl chloride (2.1 g, mol, 0.011mol,, 1.1 equiv.), Et3N (1.53 mL, 0.011 mol, 1.1 equiv.) and CH2Cl2 (mL), the desired aryl tosylate (287) was isolated after a DCM workup a white powder (1.95 g, 62 %). The spectral data were in accordance with those reported in literature.468 1 H NMR (500 MHz, CDCl3) δ 7.71 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.54 – 7.49 (m, 1H, Ar-H), 7.44 (t, J = 8.0 Hz, 1H, Ar-H), 7.33 (dd, J = 8.6, 0.6 Hz, 2H, 2 x Ar-H), 7.23 (d,
J = 8.5 Hz, 1H, Ar-H), 7.19 – 7.17 (m, 1H, Ar-H), 2.46 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 149.74 (Ar-C), 146.07 (Ar-C), 132.25 (q, J = 33.3 Hz,
CCF3), 132.01 (Ar-C), 130.45 (Ar-CH), 130.06 (Ar-C), 128.68 (Ar-CH), 126.18 (CF3),
124.03 (q, J = 3.8 Hz, Ar-CH), 119.89 (q, J = 3.8 Hz, Ar-CH), 21.86 (s, CH3).
190 | Chapter 10: Experimentals
10.4.3.7 4-Nitrophenyl 4-methylbenzenesulfonate (288)
Following the general procedure, using 4-nitrophenol (1.39 g, 0.01 mol), p-tosyl chloride (2.1 g, 0.011mol, 1.1 equiv.), Et3N (1.53 mL, 0.011 mol, 1.1 equiv.) and
CH2Cl2 (15 mL), the desired aryl tosylate (288) was isolated after a DCM workup and recrystallisation (CH2Cl2) as light orange crystals (2.04 g, 72 %). The spectral data were in accordance with those reported in literature.468 1 H NMR (500 MHz, CDCl3) δ 8.18 (d, J = 9.2 Hz, 2H, 2 x Ar-H), 7.72 (d, J = 8.4 Hz, 2H, 2 x Ar-H), 7.35 (d, J = 8.0 Hz, 2H, 2 x Ar-H), 7.18 (d, J = 9.2 Hz, 2H, 2 x Ar-H),
2.46 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 154.04 (Ar-C), 146.37 (Ar-C), 131.86 (Ar-C), 130.23
(Ar-C), 128.61 (Ar-CH), 125.52 (Ar-CH), 123.35 (Ar-CH), 21.90 (CH3).
10.4.3.8 Mesityl 4-methylbenzenesulfonate (289)
Following the general procedure, using 2,4,6-trimethylphenol (0.8 g, 5 mmol), p-tosyl chloride (1.05 g, 5.5 mmol, 1.1 equiv.), Et3N (0.72 mL, 5.5 mmol, 1.1 equiv.) and
CH2Cl2 (8 mL), the desired aryl tosylate (289) was isolated after a DCM workup and recrystallisation (CH2Cl2) as white crystals (0.22 g, 13 %). The spectral data were in accordance with those reported in literature.468 1 H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.35 (d, J = 8.0 Hz,
2H, 2 x Ar-H), 6.82 (s, 2H, 2 x Ar-H), 2.47 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.08 (s, 6H,
2 x CH3). 13 C NMR (126 MHz, CDCl3) δ 145.52 (Ar-C), 145.16 (Ar-C), 136.41 (Ar-C), 134.62
(Ar-C), 131.88 (Ar-CH), 129.95 (Ar-CH), 129.93 (Ar-CH), 128.23 (Ar-C), 21.84 (CH3),
20.82 (CH3), 17.33 (CH3).
10.4 Experimental procedures - P a r t I I | 191
10.4.3.9 [1,1'-Biphenyl]-4-yl 4-methylbenzenesulfonate (290)
Following the general procedure, using [1,1'-biphenyl]-4-ol (1.70 g, 0.01 mol), p-tosyl chloride (2.1 g, 0.011mol, 1.1 equiv.), Et3N (1.53 mL, 0.011 mol, 1.1 equiv.) and
CH2Cl2 (15 mL), the desired aryl tosylate (290) was isolated after a DCM workup and recrystallisation (CH2Cl2) as white sparkly crystals (0.28 g, 8.5 %). The spectral data were in accordance with those reported in literature.471 1 H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.57 – 7.47 (m, 4H, 4 x Ar-H), 7.43 (t, J = 7.6 Hz, 2H, 2 x Ar-H), 7.36 (d, J = 7.4 Hz, 1H, Ar-H), 7.33 (d, J =
8.1 Hz, 2H, 2 x Ar-H), 7.05 (d, J = 8.7 Hz, 2H), 2.46 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 149.15 (Ar-C), 145.52 (Ar-C), 140.32 (Ar-C), 139.95 (Ar-C), 132.62 (Ar-CH), 129.93 (Ar-C), 129.01 (Ar-CH), 128.72 (Ar-CH), 128.39 (Ar-
CH), 127.81 (Ar-CH), 127.22 (Ar-CH), 122.81 (Ar-CH), 21.88 (CH3).
10.4.3.10 3,5-Di-tert-butylphenyl 4-methylbenzenesulfonate (291)
Following the general procedure, using 3,5-di-tert-butylphenol (2.06 g, 0.01 mol), p- tosyl chloride (2.1 g, 0.011mol, 1.1 equiv.), Et3N (1.53 mL, 0.011 mol, 1.1 equiv.) and
CH2Cl2 (15 mL), the desired aryl tosylate (291) was isolated after a DCM workup and recrystallisation (CH2Cl2) as white crystals (0.69 g, 19 %, 118 – 120 °C). 1 H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.24 (s, 1H), 6.71 (s, 2H), 2.43 (s, 3H), 1.20 (s, 18H). 13 C NMR (126 MHz, CDCl3) δ 152.61 (Ar-C), 149.64 (Ar-C), 145.20 (Ar-C), 132.77 (Ar-C), 129.65 (Ar-CH), 128.87 (Ar-CH), 120.80 (Ar-CH), 116.88 (Ar-CH), 35.03
(C(CH3)3), 31.31 (C(CH3)3), 21.76 (Ar-CH3). IR (neat): 3077, 2953, 1599, 1370 + HR-MS (EI+): C21H28O3S [M] calcd 360.1759, found 360.1752.
192 | Chapter 10: Experimentals
10.4.3.11 3-Bromophenyl 4-methylbenzenesulfonate (292)
Following the general procedure, using 3-bromophenol (2.69 mL, 0.01 mol), p-tosyl chloride (2.1 g, 0.011mol, 1.1 equiv.), Et3N (1.53 mL, 0.011 mol, 1.1 equiv.) and
CH2Cl2 (15 mL), the desired aryl tosylate (292) was isolated after a DCM workup and recrystallisation (CH2Cl2) as light pink crystals (0.62 g, 19 %). The spectral data were in accordance with those reported in literature.472 1 H NMR (500 MHz, CDCl3) δ 7.71 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.38 (m, 1H, Ar-H), 7.33 (d, J = 8.0 Hz, 2H, 2 x Ar-H), 7.20 – 7.11 (m, 2H, 2 x Ar-H), 6.93 (m, 1H, Ar-H),
2.46 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 150.03 (Ar-C), 145.88 (Ar-C), 132.16 (Ar-C), 130.76 (Ar-CH), 130.47 (Ar-CH), 130.02 (Ar-CH), 128.64 (Ar-CH), 125.99 (Ar-C), 122.56
(Ar-CH), 121.29 (Ar-CH), 21.87 (CH3).
10.4.3.12 4-Iodophenyl 4-methylbenzenesulfonate (293)
Following the general procedure, using 4-iodophenol (2.20 g, 0.01 mol), p-tosyl chloride (2.1 g, 0.011mol, 1.1 equiv.), Et3N (1.53 mL, 0.011 mol, 1.1 equiv.) and
CH2Cl2 (15 mL), the desired aryl tosylate (293) was isolated after a DCM workup and recrystallisation (CH2Cl2) as an off-white crystals (2.71 g, 73 %). The spectral data were in accordance with those reported in literature.473 1 H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 8.3 Hz, 2H, 2 x Ar-H), 7.59 (d, J = 8.9 Hz, 2H, 2 x Ar-H), 7.31 (d, J = 8.0 Hz, 2H, 2 x Ar-H), 6.73 (d, J = 8.9 Hz, 2H, 2 x Ar-H),
2.44 (s, 3H, CH3). 13 C NMR (126 MHz, CDCl3) δ 149.55 (Ar-C), 145.76 (Ar-C), 138.83 (Ar-CH), 132.12
(Ar-C), 129.99 (Ar-CH), 128.61 (Ar-CH), 124.59 (Ar-CH), 91.83 (Ar-C), 21.85 (CH3).
A p p e n d i x A - Crystallographic data of compound 135C | 193
Appendices Appendix A- Crystallographic data of compound 135C X-ray diffraction data was collected by Assoc/Prof Brian W. Skelton at 100(2) K on an Oxford Diffraction Gemini or an Oxford Diffraction Xcalibur diffractometer fitted with Mo Kα radiation. Following multi-scan absorption corrections and solution by direct methods, the structure was refined against F2 with full-matrix least-squares using the programme SHELXL-97.474 All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were added at calculated positions and refined by use of a riding model with isotropic displacement parameters based on those of the parent atoms
A1 Compound 135C
Table A1. Crystal data and structure refinement for 135C. Identification code nym314
Empirical formula C52 H62 O10 Formula weight 847.02 Temperature 100(2) K Wavelength 1.54178 Å Crystal system Monoclinic
Space group P21 Unit cell dimensions a = 6.2530(2) Å b = 32.3897(15) Å c = 22.4588(13) Å
Volume 4547.6(4) Å3 194 | A p p e n d i c e s
Z 4 Density (calculated) 1.237 Mg/m3 µ 0.682 mm-1 Crystal size 0.45 x 0.08 x 0.04 mm3 θ range for data collection 3.36 to 67.50°. Index ranges -7<=h<=7, -33<=k<=38, -26<=l<=26 Reflections collected 42750 Independent reflections 12366 [R(int) = 0.0772] Completeness to θ= 67.50° 99.0 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 1.00/0.88 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 12366 / 1 / 1123 Goodness-of-fit on F2 1.219 Final R indices [I>2σ(I)] R1 = 0.1160, wR2 = 0.2893 R indices (all data) R1 = 0.1346, wR2 = 0.3143 Largest diff. peak and hole 1.072 and -0.381 e.Å-3
Figure A1. Projection of molecule of 135C onto the plane of the central phenyl ring.
Table 2. Hydrogen bonds for 135C [Å and °]. ______A p p e n d i x A - Crystallographic data of compound 135C | 195
_____ D-H...A d(D-H) d(H...A) d(D...A) <(DHA) ______O(110)-H(110)...O(601) 0.84 1.94 2.650(7) 142.0 O(130)-H(130)...O(239) 0.84 1.83 2.637(7) 161.8 O(150)-H(150)...O(259) 0.84 1.81 2.650(8) 176.9 O(210)-H(210)...O(401) 0.84 1.79 2.615(8) 168.0 O(230)-H(230)...O(139) 0.84 1.82 2.643(8) 164.3 O(250)-H(250)...O(159) 0.84 1.82 2.653(7) 175.1 ______Hydrogen bond with another 135C Hydrogen bond with another 135C Hydrogen bond with THF 196 | Bibliography
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